Lymph node as a site for transplantation, organogenesis and function for multiple tissues and organs

ABSTRACT

The present invention relates to methods and compositions for transplanting non-lymphoid tissues into lymphoid organs. It may be used to cultivate organ tissues including for the purpose of supplementing or reconstituting organ function. Tissues that may be propagated in this manner include but are not limited to lung, kidney, thyroid, intestine, and brain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/810,064 filed Jul. 27, 2015, which is a continuation of InternationalApplication Serial No. PCT/US2014/021420 filed Mar. 6, 2014, whichclaims priority to U.S. Application Ser. No. 61/773,625 filed Mar. 6,2013; and is also a continuation-in-part of U.S. application Ser. No.12/921,001 filed Sep. 3, 2010, issued as U.S. Pat. No. 9,125,891, whichis a U.S. National Stage Patent Application under 35 U.S.C. § 371 ofInternational Application Serial No. PCT/US2009/036506 filed Mar. 9,2009, which claims priority to U.S. Application Ser. No. 61/068,548,filed Mar. 7, 2008, the contents of each of which are incorporated byreference in their entireties herein, and priority to each of which isclaimed.

GRANT INFORMATION

This invention was made with government support under grant numberDK085711 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 29, 2021, isnamed 072396 0873 SL.txt and is 3,493 bytes in size.

INTRODUCTION

The present invention relates to methods and compositions fortransplanting non-lymphoid tissues into lymphoid organs. It may be usedto cultivate organ tissues for purposes including supplementing orreconstituting organ function. Tissues that may be propagated in thismanner include but are not limited to lung, kidney, thyroid, intestine,and brain.

BACKGROUND OF THE INVENTION

The shortage of organs available for transplant to terminally illpatients represents a major worldwide medical, social and economicchallenge. An alternative approach to whole-organ transplant involvesthe transplantation of cells to regenerate failing organs (1,2).However, orthotopic cell-based therapy directed at a diseased organ maynot be feasible for many reasons, ranging from a possible lack of anappropriate environment in cirrhotic and fibrotic liver during end-stagedisease to the lack of a thymus in complete DiGeorge syndrome (3-5).Consequently, a crucial requirement of cell-based therapy for thesepatients is to establish an optimal in vivo site for cell and tissuetransplantation to restore organ functions (6,7).

The lymph node is a key organ of the mammalian immune system that hasevolved to mount an immediate and orchestrated response against invadingpathogens. The lymph node acts as a checkpoint where migrating T and Bcells may encounter foreign antigens (8,9). If a foreign antigen isidentified, T cells undergo rapid cell division and also signal for helpand recruit additional T cells (8,9). To accommodate this suddenincrease in cell number, lymphocytes need a special environment, whichthe lymph node provides.

Interestingly, the lymph node is also one of the first clinicallyobserved sites of most cancer metastasis. Selected cancer cells willoften migrate away from a primary tumor and colonize the lymph node(10). Lymphatic vessels are designed to facilitate the uptake ofsurrounding fluid and cells, which are then transported to a nearbylymph node (10). Therefore, malignant tumor cells take advantage of thisroute normally traveled by immune cells. On arrival in the lymph node,tumor cells can survive, perhaps because the architecture of the lymphnode provides direct access through the high endothelial venules toessential nutrients and growth factors found in the blood. The lymphnode also contains fibroblastic reticular cells and other stromal cellsthat secrete chemokines to enhance cell recruitment and survival(8,9,11).

SUMMARY OF THE INVENTION

The present invention relates to the use of the lymph node environmentto promote the survival and expansion of healthy cells and tissues.Healthy cell and tissue growth in lymph nodes would provide a newapproach for cell therapies in regenerative medicine.

The present invention is based, at least in part, on the discoveriesthat (i) hepatocytes injected directly into a single jejunal, popliteal,axillary or periportal lymph node generate an ectopic hepatic mass andrescue mice from lethal liver failure; (ii) thymic tissue injected intosingle jejunal lymph nodes of athymic nude mice generates functionalectopic thymuses; (iii) pancreatic islets transplanted into singlejejunal lymph nodes of streptozotocin-induced diabetic mice engraft andsecrete insulin to normalize glucose concentrations; (iv) additionalfetal tissues including brain, lung, intestine, kidney and thyroid couldalso be successful engrafted into lymph nodes, and in various casesproduced a histologic resemblance to the native organ.

In certain embodiments, the present invention provides for methods ofinducing tolerance in a subject to transplantation of allograft tissue.In certain embodiments, the method of inducing tolerance comprisesconditioning a transplant recipient with cells immune-matched to a donorof a subsequent allograft. In certain embodiments, the immune-matchedcells are thymic cells. In certain embodiments, the thymic cells aretransplanted into the lymph node of the recipient.

In certain embodiments, the method of inducing tolerance comprisesincreasing regulatory T cell (Treg) induction associated with cross-talkbetween donor thymus tissue and recipient thymus tissue. In certainembodiments, the Treg cells are CD4+, CD25+ and/or FoxP3+ Treg cells.

In certain embodiments, the present invention also provides a method oftransplanting allograft tissue to a subject comprising (i) introducingnon-lymphoid cells in a lymphoid tissue of the subject under conditionssuch that the cells are able to proliferate; and (ii) introducingallograft tissue to the subject after the non-lymphoid cells have beenintroduced into the lymphoid tissue of the subject.

In certain embodiments, the non-lymphoid cells are immune-matched to theallograft tissue.

In certain embodiments, the non-lymphoid cells are thymus cells.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E Direct injection of hepatocytes into a single lymph node ofa C57BL/6 wild-type mouse. (FIG. 1A) Jejunal lymph node (LN, yellowdotted oval) not transplanted (top) and just after transplantation(bottom) with primary hepatocytes, which were mixed with 3% Evans bluedye and Matrigel before injection. Scale bars, 1 mm. (FIG. 1B) In vivooptical imaging of mice into which primary hepatocytes from luciferasetransgenic mice were injected into a single jejunal or popliteal lymphnode, into the spleen (SP) or intraperitoneally (IP). Signals (blue tored) depict different concentrations of donor hepatocytes at day 1 (top)and 1 week (bottom) after transplant. (FIG. 1C Top left, whole-mountimaging of a jejunal lymph node 1 week after injection of donor GFP+hepatocytes. Shown is the bright-field image merged with thefluorescence. Top middle to bottom right, immunofluorescent staining offrozen lymph node serial sections with monoclonal antibodies (mAbs)(red) to ER-TR7 (reticular fibroblasts), LYVE-1 (lymphatic vessels),PNAd (high endothelial venules), B220 (B cells) and CD4/CD8 (CD4 T andCD8 T cells) with the presence of GFP+ hepatocytes (green). Dotted linesindicate the lymph node boundary. Scale bars, 100 μm. (FIG. 1D)Immunofluorescent staining of donor GFP+ hepatocytes (green) in jejunallymph nodes 1 week after transplantation. Shown are serial sectionsstained with mAbs (red) to E-cadherin (E-Cad), C—C chemokine receptortype 7 (CCR7) and S1PR1, as well as native liver sections stained ascontrols (right). In native liver, CCR7 (red) was co-stained withdipeptidyl peptidase-4 (DPPIV) (green). All sections were counterstainedwith Hoechst 33342 (blue). Scale bars, 100 μm. (FIG. 15E) Proliferationof engrafted hepatocytes in lymph nodes after partial hepatectomy (PHx).Paraffin sections of injected lymph nodes and corresponding native liver1 and 2 weeks (1 W and 2 W, respectively) after transplantation stainedfor GFP (1 and 2 weeks after transplantation) and BrdU (2 weeks aftertransplantation) and revealed by peroxidase (brown cytoplasmic and brownnuclei staining, respectively). Sections were counterstained withhematoxylin. Yellow outlines mark small populations of GFP+ hepatocytes.The bar graphs show the number of GFP+ hepatocytes and the percentage ofBrdU+ hepatocytes per section after immunostaining at 2 weeks aftertransplantation in mice with or without PHx. *P<0.05, **P<0.0001. Data(mean±s.e.m.) are representative of one experiment with three to fivemice per group. The experiment was repeated twice. Scale bars, 100 μm.

FIGS. 2A-2E Direct injection of hepatocytes into a single lymph node ofa Fah−/− mouse. (FIG. 2A) Macroscopic appearances of nontransplanted andtransplanted (hepatized) lymph nodes (lymph nodes are marked by yellowdotted ovals). For nontransplanted axillary and popliteal lymph nodes,the presence of the lymph node is highlighted by injected Evans blue.Shown are representative images of the jejunal, axillary and poplitealhepatized lymph nodes captured at 12, 22 and 25 weeks aftertransplantation, respectively. Scale bars, 5 mm. (FIG. 2B) Histology ofhepatized jejunal lymph nodes in rescued Fah−/− mice. Left, serialsections of hepatized lymph nodes stained with hematoxylin and eosin(H&E) (top left), Fah (brown) and counterstained with hematoxylin (blue)(middle left). The immunofluorescence shows lymphatic vessels (LYVE1,red) and hepatocytes (GFP+, green) (bottom left). Right, highermagnification of H&E staining showing typical cuboidal hepatocytes.Reticular fibroblasts stain with antibodies specific for ER-TR7 (red),whereas hepatocytes are GFP+ and stain with antibodies specific forDPPIV (red). Glutamine synthetase (GS, red) expression shows uniquezonal restriction surrounding terminal hepatic venules. Sections werecounterstained with Hoechst 33342 (blue). Scale bars, left, 1 mm; middleand right, 100 μm. (FIG. 2C) Kaplan-Meier survival curves (top) and bodyweight (bottom) of Fah−/− mice transplanted in intra- andextra-abdominal lymph nodes compared to mice given no treatment. Errorbars show the standard error. (FIG. 2D) Left, macroscopic appearances ofnormal and hepatized lymph nodes (yellow dotted ovals) in the periportalarea. Top right, Kaplan-Meier survival curves of Fah−/− micetransplanted into the jejunal or periportal lymph node. Bottom right,average weights of periportal and jejunal lymph nodes and intra-(intra-abd) and extra-abdominal (extra-abd) lymph nodes. No statisticaldifference (NS) was observed. Error bars show s.e.m. Scale bars, 5 mm.(FIG. 2E) Top, lymph node injection (LN-Tx) and splenic injection(SP-Tx) of GFP+ hepatocytes (C57BL/6 background) into Fah−/− mice (129svbackground) after removal of2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) (lackof NTBC in drinking water induces liver failure in Fah−/− mice). Whereindicated, mice were injected with the immunosuppressive agents CTLA4-Igand MR1 on days 0, 2, 4 and 6 after transplantation. The experiment wasrepeated twice. The images n the bottom show one representative LN-Txmouse injected with CTLA4-Ig and MR1. Also shown are the macroscopicappearances of hepatized jejunal lymph nodes (yellow dotted ovals) andimmunostaining of the hepatized lymph nodes for the presence of GFP+(green) and Fah+ (red) hepatocytes, as well as reticular fibroblasts(ER-TR7, red). Sections were counterstained with Hoechst 33342 (blue).Scale bars, left, 5 mm; middle and right, 100 μm.

FIGS. 3A-3E Functional ectopic thymus in the jejunal lymph node. (FIG.3A) Flow cytometric analysis of T cells in the peripheral blood. Top,representative analysis of the gating strategy for CD4 and CD8 T cellsin wild-type C57BL/6 (WT), BALB/c nude (Nude) and BALB/c nude mice intowhich C57BL/6 GFP+ thymic cells were injected into a jejunal lymph node(LN-Tx nude). The number values assigned to the gates and quadrantsrepresent the percentage of total live cells within that gate orquadrant. All contour plots display 10% probability contours. Bottom,the percentage of CD3+, CD3+CD4+ and CD3+CD8+ live T cells in the bloodof each mouse analyzed (each symbol represents one mouse); the mice wereWT, nude, KC-Tx nude (BALB/c nude mice transplanted under the kidneycapsule) and LN-Tx nude. The thin black line indicates the mean±s.e.m.No statistical significance (NS) was observed between kidney capsule andlymph node transplantation in each of the three groups. (FIG. 3B) Left,representative flow cytometric analysis of cells present in wild-typeC57BL/6 (WT) and LN-Tx nude mouse tissues. The number values assigned tothe gates and quadrants represent the percentage of total live cellswithin that gate or quadrant. Bottom, whole-mount jejunal lymph node ofa nude mouse engrafted in the lymph node with GFP+ thymic cells (topleft). The bright-field image was merged with the fluorescence. Topright, a frozen section with GFP+ donor thymic cells. Bottom right,immunostaining of cytokeratin 5 (K5) (blue) and cytokeratin 8 (K8) (red)and counterstaining with Hoechst (white). Bottom left, WT native thymusstained for K5 (red) and K8 (green). Scale bars, 200 μm. (FIG. 3C) Flowcytometric analysis of TCR Vβ segment expression in splenocytes fromwild-type C57BL/6 mice (WT C57BL/6), heterozygous BALB/c nude mice (Hetnude) or LN-Tx mice. The bar graphs show the mean percentage of theparticular Vβ receptor. Individual symbols represent data from a singlemouse (n=4-5). (FIG. 3D) Dot plots and histograms show the gatingstrategy to detect regulatory T cells (FoxP3+ fraction of CD4+CD25+ Tcells) and naive (CD44−CD62L+), central memory (CD44+CD62L+) andeffector memory (CD44+CD62L−) T cells in splenocytes. Graphs show thedata from individual mice, labeled as in panel 3C. The thin black lineindicates the mean. (FIG. 3E) Top, Kaplan-Meier curves showing thesurvival of skin grafts from C57BL/6 or CBA/CaJ donor mice transplantedonto BALB/c nude recipients that had previously undergonetransplantation of C57BL/6 thymic cells into the lymph node (LN-Tx nude)or into unmanipulated nude mice. Middle, graph of tumor growth inathymic BALB/c nude (nu/nu) mice. Values are mean±s.e.m. n=10. Bottom,presence (+) or absence (−) of tumor growth after a single subcutaneousinjection of 300,000 human colorectal cancer cells into LN-Tx nude,BALB/c nude (Nude) or C57BL/6 wild-type (WT) recipients.

FIGS. 4A-4C Ectopic pancreas generation in the jejunal lymph node afterislet transplantation. (FIG. 4A) Top, whole-mount lymph node of astreptozotocin-treated diabetic C57BL/6 mouse engrafted with C57BL/6GFP+ pancreatic islets. The bright-field image was merged with thefluorescence. Other images show immunofluorescence of lymph nodesremoved 6 weeks after engraftment. Staining with antibodies specific forERTR7 (reticular fibroblasts), C-peptide (C-PEP) and glucagon (GLUC) isshown in red, GFP is shown in green, and Hoechst counterstain is shownin blue. (FIG. 4) Average blood glucose concentrations in diabeticrecipient mice over the course of 10 weeks after transplantation ofislets into the jejunal lymph nodes (LN-Tx, n=5), under the kidneycapsule (KC-Tx, n=3) or in diabetic mice with no transplantation (No Tx,n=6). The data are presented as means±s.e.m. (FIG. 4) Average bodyweight (left) and blood glucose concentrations (middle) of C57BL/6wild-type (WT C57) mice or C57BL/6 LN-Tx mice after LPS injection (1 mgper kg of body weight). Right, average serum concentrations of TNF-α,IL-1β and IL-6 2 h after LPS injection. All error bars show standarderror. All immunofluorescent image scale bars are 100 μm unlessotherwise indicated.

FIGS. 5A-5B. Neovascularization of ectopic tissue. (FIG. 5A) Vasculartrees are shown in a native lymph node and native liver of a GFPtransgenic mouse and in mice after hepatocyte transplantation into thelymph node. (FIG. 5B) Immunostaining of lymph nodes injected withhepatocytes, thymic cells and pancreatic islets. Images were captured12, 15 and 6 weeks after the transplant of each tissue, respectively.CD31 is a marker for blood vessels, and CD105 and Collagen IV aremarkers for neovascularization. Vasculature markers are shown in red,and ectopic tissue is shown in green. All sections were counterstainedwith Hoechst 33342 in blue. All scale bars are 100 μm.

FIG. 6. Distribution of immune cells in the hepatized jejunal LN of arescued C57BL/6 Fah−/− mice at 12 weeks after transplantation. Left toright panels, immunostaining of frozen LN serial sections with mAbs(red) against CD4/CD8 (CD4 and CD8 T cells), Gr-1 (granulocytes) andF4/80 (macrophages) with the presence of C57BL/6 GFP+ hepatocytes(green). All sections were counterstained with Hoechst 33342 (blue).Scale bar: 100 mm.

FIG. 7. Representative flow cytometry analysis of peripheral blood Tcells. Analysis of CD4 and CD8 T cells from C57BL/6 GFP+ mice, wild typeC57BL/6, BALB/c Nude, kidney capsule (KC) transplanted (Tx) BALB/c Nude,and lymph node (LN) transplanted BALB/c Nude mice. The number valuesassigned to the gates and quadrants represent the percentage of totallive cells within that gate or quadrant. All contour plots display 10%probability contours.

FIGS. 8A-8B. Presence of peripheral T cells 10 months after C57BL/6 GFP+thymic transplantation in BALB/c Nude LN. (FIG. 8A) Blood analysis of 3mice; the number values assigned represent the percentage of total liveCD4 and CD8 T cells. (FIG. 8B) Flow cytometric analysis of mouse L084.The number values assigned to the gates and quadrants represent thepercentage of total live cells within that gate or quadrant. All contourplots display 10% probability contours.

FIG. 9. Table 1.

FIGS. 10A-10B. (FIG. 10A) C57BL/6 GFP+ thymic tissue engrafts in thesubcapsular space of the jejunal BALB/c Nude LN. Frozen section of anectopic thymus with GFP+ donor thymic cells and stained with cytokeratin8 (K8) in red. Hoechst counterstain is shown in blue. Scale bar: 100 mm.(FIG. 10B) Engrafted C57BL/6 GFP+ islet vasculature is derived from therecipient C57BL/6 mouse 6 weeks after transplantation. Immunostainingagainst CD31 (red) does not co-stain with the GFP+ donor derived islets(green). Scale bar: 50 mm.

FIGS. 11A-11B. (FIG. 11A) Harvest of the uterine horns from a pregnantmouse, and removal of placenta and fetal membranes from an embryo.Sagittal and transversal paraffin sections of an embryo stained withHematoxylin and Eosin. (FIG. 11B) Scheme of the jejunal lymph nodeinjection procedure using different mouse fetal tissues.

FIGS. 12A-12D. Each panel shows paraffin (FIGS. 12A, 12B and 12D) orfrozen (FIG. 12C) sections of donor C57BL/6 GFP+ tissues stained withHematoxylin and Eosin or Hoechst, respectively, whole-mount jejunallymph nodes of C57BL/6 mice 3 weeks after transplantation, andimmunofluorescence staining of frozen lymph node serial sections withthe presence of GFP+ cells.

FIG. 13. (Upper) Dissection of mouse thyroid gland. (Bottom) Scheme ofthe jejunal lymph node injection procedure, whole-mount jejunal lymphnode 3 weeks after transplantation, and immunofluorescence staining of afrozen lymph node section with the presence of GFP+ cells.

FIGS. 14A-14C. Problems associated with determining gestational age.

FIG. 15. Table showing mice injected and lymph nodes repopulated forvarious organ types.

FIG. 16. Transplantation of thyroid gland into lymph node.

FIG. 17. Transplantation of liver into lymph node.

FIG. 18. Transplantation of brain tissue into lymph node.

FIG. 19. Transplantation of lung tissue into lymph node.

FIG. 20. Transplantation of intestinal tissue into lymph node.

FIGS. 21A-21C. Transplantation of kidney tissue into lymph node.

FIGS. 22A-22D. Transplantation of kidney tissue into lymph node.

FIGS. 23A-23D. The lymph node is a permissive site for kidneyorganogenesis. (FIG. 23A) Schematic view of kidney transplantation intothe lymph node. (FIG. 23B) Hematoxylin and Eosin (H&E) staining of aparaffin section of donor C57BL/6 GFP+ embryonic kidney showing S-shapedbodies (upper left); whole-mount jejunal lymph node of a C57BL/6 mouse 3weeks after embryonic kidney transplantation (upper right), and pictureof a frozen lymph node section stained for reticular fibroblasts andreticular fibers (ER-TR7), with the presence of GFP+ cells (lower).Nuclei were counterstained using Hoechst. (FIG. 23C) Picture of a frozenlymph node section with the presence of GFP+ cells (upper). Enlargedviews of the collagen IV-stained boxed regions are shown (lower). Nucleiwere counterstained using Hoechst. (FIG. 23D) Immunofluorescencestaining for CD31, podoplanin, claudin-2, keratin-8, and erythropoietin(Epo) of frozen sections of a 3-week repopulated lymph node with thepresence of GFP+ cells. Nuclei were counterstained using Hoechst.

FIGS. 24A-24C. Proliferative and urine-concentrating ability of 6-weekectopic grafts. (FIG. 24A) GFP positivity (left) and H&E staining(right) of a frozen or paraffin section of a jejunal lymph node 6 weeksafter transplantation. (FIG. 24B) Picture of paraffin lymph nodesections stained for GFP or BrdU (AEC, red color). (FIG. 24C)Representative RT-PCR analysis for different urea transporters anderythropoietin (Epo) in lymph node 6 weeks after embryonic kidneyinjection as compared to a control lymph node.

FIG. 25. Host cells vascularize the developing tissue.Immunofluorescence staining for podoplanin, CD31, collagen IV,keratin-8, and erythropoietin of lymph node frozen sections with thepresence of GFP+ cells. Nuclei were counterstained using Hoechst.

FIGS. 26A-26F. Renal cyst development in repopulated lymph nodes. (FIG.26A) Whole-mount jejunal lymph node of a C57BL/6 mouse 12 weeks afterembryonic kidney transplantation showing GFP positivity (left), andhematoxylin and eosin (H&E) staining showing cysts (right). (FIG. 26B)Detail of cyst #1 epithelium stained with H&E, periodic acidschiff(PAS), masson's trichrome (TRI), picro-sirius red (PSR), GFP, BrdU,aquaporin-1 (AQP1), and sodium-potassium-chloride transporter 2 (NKCC2)(left), and of cyst #2-3 epithelium stained with H&E, PAS, TRI, PSR,GFP, BrdU, AQP1 and 2 (right, yellow arrows indicates vacuoles). (FIG.26C) Details of proteinaceous material and fibers found inside cyst #1(left) and of round globules found inside cyst #2 and 3 (right), afterstaining with H&E, PAS, TRI, and PSR. (FIG. 26D) Pictures of urinarycrystals found inside cyst #1 (left), and Blood Urea Nitrogen (BUN)levels in serum and lymph node fluid of a transplanted mouse versus acontrol mouse (right). (FIG. 26E) Detail of cyst #3 epithelium stainedwith GFP. (FIG. 26F) Details of repopulated lymph node stained with H&E,PAS, TRI, and PSR, BrdU, or collagen IV showing glomerular and tubularalterations (black arrows indicate BrdU+ nuclei; yellow arrows indicatesvacuoles).

FIGS. 27A-27D. Bone marrow-derived host cells contribute to mesangialcells and podocyte regeneration. (FIG. 27A) Fluorescence intensityprofiles of GFP expressing leukocytes in peripheral blood of bone marrowchimeric mice. Blood of a wild type and a GFP+ mouse were used asnegative and positive control, respectively. (FIG. 27B) Overview ofexperimental plan (IR, irradiation; EK, embryonic kidney; LN, lymphnode). (FIG. 27C) Representative ectopic glomerulus grown inside lymphnodes of bone marrow chimeric mice, showing bone marrow-derived cellcontribution to glomerular mesangium. Sections were stained withcollagen IV antibody and nuclei were counterstained using Hoechst. (FIG.27D) (left) I-III, pictures of a lymph node section from bone marrowchimeric mouse showing localization of bone marrow-derived cells in thekidney graft; IV-VI, enlarged view of pictures I-III. (right)Representative ectopic glomeruli as in FIG. 27C. Sections were stainedwith collagen IV, CD45, CD106, CD3, CD4, CD8, CD45R/B220, Ly6C/G, F4/80,CD31, podoplanin, WT-1 or Epcam. Nuclei were counterstained usingHoechst. Insets show the presence of GFP+ CD45−, GFP+ CD106+, or GFP+WT1+ cell subsets inside the ectopic glomeruli.

FIGS. 28A-28C. Nephrectomy accelerates kidney organogenesis anddegeneration. (FIG. 28A) Overview of experimental plan (EK, embryonickidney; Nx, nephrectomy; LN, lymph node; K, kidney; see Materials andMethods section for details). (FIG. 28B) Renal cell proliferation shownby bromodeoxyuridine (BrdU) incorporation. (FIG. 28C) Representativeectopic grafts of nephrectomized mice and sham-operated controls (upper)as compared to 3 or 12 weeks ectopic grafts (bottom).

FIG. 29. Middle Panel: Schematic view of transplantation of E14.5-15.5or P3 kidney into the lymph node, and immunofluorescence staining forpodoplanin of an adult kidney section isolated from a GFP+ mouse(lower). Left and Right Panels, from the top to the bottom:immunofluorescence staining for podoplanin of frozen sections ofembryonic (left) or new born (right) kidney showing different maturity;whole-mount mouse jejunal lymph node 3 weeks after embryonic (left) ornew born (right) kidney transplantation, showing different engraftment;immunofluorescence staining for podoplanin of lymph node frozen sections3 weeks after transplantation of embryonic (left) or new born (right)kidney. Nuclei were counterstained using Hoechst.

FIGS. 30A-30C. (FIG. 30A) Gating strategy for fluorescence-activatedcell sorting (FACS) analysis of peripheral blood of chimeric mice 6weeks after bone marrow transplant. Blood cells were gated on livecells, singlets, leukocytes (upper). Both GFP+ and GFP− leukocytesubsets were analyzed for CD3, CD19/CD45R or CD11b/Ly6G−LY6C (middle).The CD3+ cell population was further analyzed for CD4/CD8 (lower). (FIG.30B) Dot plot graph showing percentages of different GFP+ leukocytepopulations from bone marrow chimeric mice. (FIG. 30C) Stacked bar graphshowing percentages of donor GFP+/Ly6G-−Y6C+ versus host GFP−/Ly6G−LY6C+cells. Cells were gated on live, Ly6G−LY6C, GFP. A representativehistogram profile is shown. Blood from a wild type and a GFP+ mouse wereused as control.

FIG. 31. Representative ectopic glomerulus grown inside lymph nodes ofbone marrow chimeric mice 10 weeks after transplantation showing nodularlesion. Sections were stained with claudin-2, WT-1, podoplanin, CD31,keratin-8, and vimentin. Nuclei were counterstained using Hoechst.

FIGS. 32A-32C. Astrogenesis in the developing ectopic brain. (FIG. 32A)Schematic view of transplantation of multiple embryonic tissues into thelymph node (scale bar, 1 mm). (FIG. 32B) Table shows percentages ofengraftment into the mouse lymph node for different tissues. (FIG. 32C1)Hematoxylin and Eosin (H&E) staining of a paraffin section of donorembryonic brain (upper left); whole-mount jejunal lymph node 3 weeksafter embryonic brain transplantation (GB3 LN, upper right), andpictures of frozen lymph node (GB3 and GB4 LN) sections with thepresence of GFP+ cells (lower). Nuclei were counterstained usingHoechst. (FIG. 32C2) Mouse embryonic brain transversal section (upper),and pictures of GB3 and GB4 LN sections stained for GFAδ with thepresence of GFP+ cells (lower). Nuclei were counterstained usingHoechst.

FIGS. 33A-33D. Granulocyte/macrophage progenitor accumulation followingembryonic thymus transplantation into the lymph node (LN), and hostcontribution in the generation of the ectopic thymic cortex. (FIG. 33A)Gating strategy for FACS analysis of peripheral blood of mice (M1 -M5)receiving thymus transplant into their lymph nodes. Blood cells weregated on live cells, leukocytes, singlets, granulocyes/myeloid cells orlymphocytes. (FIG. 33B) Representative fluorescence intensity histogramsof granulocyes/myeloid cells from M2 analyzed for Ly6G−Ly6C (upper) orCD11b (lower) at 0, 3, 6, 12, or 21 weeks after thymus transplant. (FIG.32C) Representative flow cytometric contour plots of granulocyes/myeloidcells from M2 stained for Ly6G−Ly6C and CD11b, and gated onCD11b+/Ly6G−Ly6C−/low, CD11b+/Ly6G−Ly6Cint, and CD11b+/Ly6G−Ly6Chigh at0, 3, 6, 12, or 21 weeks after thymus transplant. (FIG. 32D) Dot plotsshowing frequency of CD11b+/Ly6G−Ly6C−/low, CD11b+/Ly6G-−y6Cint, andCD11b+/Ly6G−Ly6Chigh at 0, 3, 6, 12, or 21 weeks after thymustransplant. Each symbol represents one mouse, and the horizontal barsrepresent the median values. *p<0.05, **p<0.01, ***p<0.001.

FIGS. 34A-34C. Contribution of the host in the generation of the ectopicthymic cortex. (FIG. 34A) Agarose gel electrophoresis of PCR productsfollowing semi-quantitative RT-PCR analysis for GM-CSF (expectedamplicon size of 431 bp) in embryonic thymus (EmT), 6- (6 wEcT) or21-week ectopic thymus (21 wEcT), and adult thymus (AdT). Wild typelymph node (LN) was used as a negative control. Amplification of GAPDHwas used as an internal control. The densitometric scanning data fromtwo experiments are shown as bar graphs of GM-CSF/GAPDH ratio on theright (6 wEcTs were isolated from M4 and M5, while 21 wEcTs wereisolated from M1 and M3). (FIG. 34B) Picture of thymus glands isolatedfrom a C57BL/6 GFP+ embryo (upper left) and H&E staining of a paraffinsection of embryonic thymus (EmT, upper right); whole-mount mousejejunal lymph nodes 21 weeks after embryonic thymus transplantation,showing different engraftment (21 wEcT, lower). (FIG. 34C)Immunofluorescence staining for keratin 8 (K8) or keratin 5 (K5) of21-week ectopic thymus from M2 with the presence of GFP+ cells (left).Nuclei were counterstained using Hoechst. Enlargements of K8 and K5stainings are shown on the right, together with stainings of GFP, CD31,and CD105 (CD31 and CD105 pictures were taken from a lymph node isolatedfrom M6).

FIGS. 35A-35C. Presence of terminally differentiated, mucus-producingcells in ectopic lung, stomach and intestine tissues. (FIG. 35A-35C).Each panel shows H&E staining of a paraffin section of donor embryoniclung (EmL), stomach (EmS) or intestine (EmI); whole-mount jejunal lymphnode 3 weeks after transplantation of embryonic lung (3 wEcL), stomach(3 wEcS) or intestine (3 wEcI), and pictures of frozen lymph nodesections stained with specific markers with the presence of GFP+ cells.Nuclei were counterstained using Hoechst (ER-TR7, Reticular Fibroblastsand Reticular Fibres; CgA, chromogranin A).

FIG. 36. Ectopic liver, pancreas and thymus has been successfully grownin lymph node tissue.

FIG. 37. Experimental plan for assessing the utility of transplantedthymus in mediating acceptance of allografts. 129 Fah−/− mice weretransplanted with neonatal (d2-4) Balb/c GFP thymus in the lymph node(LN). An immunosuppression regiment consisting of MR-1 and rapamycin wasstarted concomitantly. 6 weeks after thymus transplant, mice were eithergiven skin grafts or hepatocyte transfers to assess ability of thymus tomediate acceptance of subsequent allogeneic grafts. Mice receiving justimmunosuppression (IS controls) were used as controls.

FIGS. 38A-38C. (FIG. 38A) Presence of donor-specific GFP+ cells in thelymph node (LN) of 129sv mice transplanted with Balb/c-GFP neonatalthymus in the lymph node. (FIG. 38B) GFP+ cells in thymus-transplantedLN were positive for CD11c and EpCam, markers of thymic dendritic cells(DCs) and epithelial cells, respectively. (FIG. 38C) Thymus-transplantedLN showed presence of CD4/CD8 double-positive (DP) T cells (a marker ofT cell development), similar to a regular thymus. No presence of DP Tcells in a non-transplanted LN.

FIGS. 39A-39D. Long-term acceptance of allogeneic hepatocytes mediatedby ectopic thymus in the lymph node. (FIG. 39A) C57BL6/J and Balb/c skingrafts in 129.Fah−/− mice receiving Balb/c thymus (FIG. 39B) Rescue ofliver failure as evidenced by weight gain and survival in Balb/cthymus-transplanted 129.Fah−/− mice receiving Balb/c hepatocytes. (FIG.39C) Liver in Balb/c hepatocyte transferred mice showed normal livermorphology by hematoxylin and eosin (H&E) staining (i) and positivityfor FAH by immunohistochemistry (IHC) (ii). (iii) shows the presence oftransplanted GFP+ Balb/c hepatocytes that are also positive for FAH byimmunofluorescence (IF). (FIG. 39D) Balb/c thymus-transplanted mice shownormal levels of liver enzymes in the serum after Balb/c hepatocytetransfer.

FIGS. 40A-40C. Ectopic thymus induces donor-specific tolerance andincreased incidence of Tregs in recipients. (FIG. 40A) Splenocytes fromBalb/c thymus-transplanted mice were labeled with CellTrace™ andco-cultured with naive 129.sv (top panel), C57BL6/J (middle panel) orBalb/c splenocytes (bottom panel). After 72 hours, cells were analyzedfor CellTrace™ dilution by flow cytometry. (FIG. 40B) IFNγ levels fromcell culture supernatants in (FIG. 40A). (FIG. 40C) Total percentages ofT cells, CD4+ T cells and Tregs in the blood of thymus-transplanted mice(and controls) were determined by flow cytometry.

FIGS. 41A-41C. Characterization of cells migrating from ectopic tonative thymus. (FIG. 41A) Presence of GFP+ cells in the native thymus ofmice transplanted with Balb/c mice (top panel, 4× magnification). (FIG.41B) GFP+ cells present in the native thymus were MHC-II+ (middle panel)and CD11c+ (bottom panel). (FIG. 41C) GFP+ cells in the native thymus ofmice transplanted with Balb/c-GFP thymus in the LN are observed to beinteracting with CD4+ cells (i, inset) and CD4+CD25+ cells (ii and iii,inset). Images in FIGS. 41C (i) and 41C (ii, iii) are at 10× and 40×magnification, respectively.

FIGS. 42A-42C. Migrating DCs induce Tregs capable of suppressing T cellactivation. (FIG. 42A) CD11c+ GFP+ cells from native thymus wereco-cultured with naive CD4+ thymocytes. After 72 hours, the culture wasanalyzed for proportion of CD4+CD25+ cells (Tregs) in culture. (FIG.42B) Summarized data for Treg percentages in cultures described in (FIG.42A). (FIG. 42C) Tregs generated in culture in (FIG. 42B) are efficientin suppressing CD4+ T cell proliferation, as determined by IFNγ levels.Results representative of one of three independent experiments.

FIG. 43. Model depicting cross-talk between ectopic and native thymus intransplant recipient showing migration of antigen-presenting cells whichinduce Tregs.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for methods of propagating non-lymphoidcells in a lymphoid tissue for the purpose of producing a non-lymphoidtissue and or/providing a desirable biological function.

Suitable cells include but are not limited to embryonal cells,non-embryonal cells, progenitor cells, and reprogrammed somatic cells(47). Cells may be human or non-human cells (eg non-human primate, dog,cat, pig, cow, horse, sheep, goat, mouse, rat, rabbit, etc.). Cells maybe autologous, allogeneic, or xenogeneic. Suitable cells include but arenot limited to a kidney cell or a partially differentiated kidney cellor a kidney progenitor cell, a lung cell or a partially differentiatedlung cell or a lung progenitor cell, a thyroid cell or a partiallydifferentiated thyroid cell or a thyroid precursor cell, a brain cell ora partially differentiated brain cell or a brain progenitor cell, anintestinal cell or a partially differentiated intestinal cell or anintestinal precursor cell; a thymus cell or a partially differentiatedthymus cell or a thymic progenitor cell, a pancreas cell or a partiallydifferentiated pancreas cell or a pancreas precursor cell (in theforegoing, including islet cells), a liver cell a partiallydifferentiated liver cell and a liver precursor cell, a stomach cell ora partially differentiated stomach cell or a stomach precursor cell andso forth.

Cells can be implanted into a lymph node or multiple lymph nodes. Suchmethods are intended to encompass implantation into any lymph node,including, but not limited to: abdominal lymph nodes, celiac lymphnodes, paraaortic lymph nodes, splenic hilar lymph nodes, porta hepatislymph nodes, gastric lymph nodes (left and right), gastroomental(gastroepiploic) lymph nodes (left and right), retroperitoneal lymphnodes, pyloric lymph nodes (suprapyloric, subpyloric, retropyloric),pancreatic lymph nodes (superior pancreatic, inferior pancreatic,splenic lineal lymph nodes), hepatic lymph nodes (cystic,foraminal—including foramen of Winslow), pancreaticoduodenal lymph nodes(superior pancreaticoduodenal, inferior pancreaticodoudenal), superiormesenteric lymph nodes, ileocolic lymph nodes, prececal lymph nodes,retrocecal lymph nodes, appendicular lymph nodes, mesocolic lymph nodes(paracolic, left colic, middle colic, right colic, inferior mesentericlymph nodes, sigmoid, superior rectal), common iliac lymph nodes (medialcommon ilic, intermediate common iliac, lateral common iliac, subaorticcommon iliac, common iliac nodes of promontory), and external iliaclymph nodes (medial external iliac, intermediate external iliac, lateralexternal iliac, medial lacunar—femoral, intermediate lacunar—femoral,lateral lacunar—femoral, interiliac external iliac, obturator—externaliliac obturatory). In certain embodiments, the cells are injected into arecipient's lymph node from a donor's tissue. In certain embodiments, itis important for the lymph node to be able to swell as the graftexpands, and thus lymph nodes that are present in the peritoneal cavityare particularly useful, especially where the lymph nodes are notclosely associated with arteries or large veins.

In certain embodiments, the present invention provides for a method ofpropagating non-lymphoid cells in a lymphoid tissue for the purpose ofproducing a non-lymphoid tissue and or/providing or supplementing adesirable biological function, comprising introducing, into a lymph nodein a host, non-lymphoid cells, under conditions such that the cells areable to proliferate.

The host may be a human or non-human (eg non-human primate, dog, cat,pig, cow, horse, sheep, goat, mouse, rat, rabbit, etc).

In one particular embodiment, the present invention provides for amethod of providing or supplementing a biological function in a hostcomprising introducing, into a lymph node of a host, a non-lymphoid cellof an organ where the cell or organ normally provides said function; forexample, but without limitation, introducing an islet cell to provideinsulin production, or, introducing a thyroid cell to provide thyroxin,or introducing a kidney cell to provide kidney function (filtering,etc.), or introducing a thymus cell to provide immune function, etc.

In certain embodiments, the present invention provides for methods ofinducing tolerance in a subject to transplantation of allograft tissue.

In certain embodiments, the method of inducing tolerance comprisesconditioning a transplant recipient with cells immune-matched to a donorof a subsequent allograft. In certain embodiments, the immune-matchedcells are thymic cells. In certain embodiments, the thymic cells aretransplanted into the lymph node of the recipient.

In certain embodiments, the allograft tissue comprises a liver cell ortissue. In certain embodiments, the allograft tissue comprises a skincell or skin tissue.

In certain embodiments, the method of inducing tolerance comprisesincreasing regulatory T cell (Treg) induction associated with cross-talkbetween donor thymus tissue and recipient thymus tissue. In certainembodiments, the Treg cells are CD4+, CD25+ and/or FoxP3+ Treg cells.

In certain embodiments, the present invention also provides a method oftransplanting allograft tissue to a subject comprising (i) introducingnon-lymphoid cells in a lymphoid tissue of the subject under conditionssuch that the cells are able to proliferate; and (ii) introducingallograft tissue to the subject after the non-lymphoid cells have beenintroduced into the lymphoid tissue of the subject.

In certain embodiments, the non-lymphoid cells are immune-matched to theallograft tissue.

In certain embodiments, the non-lymphoid cells are thymus cells.

EXAMPLE 1: THE MOUSE LYMPH NODE AS AN ECTOPIC TRANSPLANTATION SITE FORMULTIPLE TISSUES Materials And Methods Mice and Tissues

Donor 129sv mice and recipient 129sv Fah−/− mice were a kind gift fromM. Grompe (Oregon Health and Science University). For allogeneicexperiments, donor hepatocytes were isolated from C57BL/6 GFP+ mice (GFPtransgene under the control of the human ubiquitin C promoter,C57BL/6Tg(UBC-GFP)30Schaa, Jackson Laboratory) and transplanted into129sv Fah−/− mice. For syngeneic experiments using C57BL/6 GFP+hepatocytes, 129sv Fah−/− mice were backcrossed for more than eightgenerations with C57BL/6 mice (Jackson Laboratory) to generate C57BL/6Fah−/− mice. Luciferase C57BL/6 transgenic mice (Luc+) expressingfirefly luciferase under the control of the broadly expressed (3-actinpromoter were kindly provided by S. Thorne (University of Pittsburgh).Donor primary hepatocytes were isolated from adult (5- to 8-week-old)mice. Donors and recipients were not matched according to gender.Newborn (1- to 3-day-old) C57BL/6 GFP+ mice were used as donors ofthymic cells. Athymic BALB/c nude Foxn1nu (Harlan) mice were used asrecipients of thymic cells. Blood collection (100 p1) was performedusing the submandibular bleeding technique. Adult (5- to 8-week-old)C57BL/6 GFP+ mice were used as the donors of pancreatic islets. Adult(5- to 8-week-old) C57BL/6 mice were used as recipients of pancreaticislets. Mice were bred and housed in the Division of Laboratory AnimalResources facility at the University of Pittsburgh Center forBiotechnology and Bioengineering. Experimental protocols followed USNational Institutes of Health guidelines for animal care and wereapproved by the Institutional Animal Care and Use Committee at theUniversity of Pittsburgh.

Hepatocyte Transplantation

Primary hepatocytes were isolated using the two-step collagenaseperfusion technique. The number and viability of the cells weredetermined by trypan blue exclusion. For each recipient, 100,000-500,000viable cells were suspended in 20 μl Matrigel (BD Biosciences) and kepton ice until transplantation. Recipient mice were anesthetized with 1-3%isoflurane and laparotomized. The jejunal lymph node was exposed, andcells were injected using a 28 G needle under a dissecting scope(Leica). Just after injection, the contact site was clipped for 5 min bymicro clamp to prevent cell leakage. In the experiments with Fah−/−mice, the mice were kept on NTBC-containing drinking water at aconcentration of 8 mg/l until transplantation. NTBC was discontinuedjust after surgery. For extra-abdominal lymph node injections, weinjected 3% Evans blue solution intradermally into the footpad of thehindlimb or forelimb before cell transplant to visualize the smallpopliteal or axillary lymph node.

Thymic Transplantation

Thymuses were harvested from newborn GFP+ transgenic mice and cut intosmall fragments. The jejunal lymph nodes were exposed, and cells wereinjected with minced thymus tissue through a 20 G needle. Thymus tissuewas also grafted beneath the kidney capsule as a positive control. Forthe kidney capsule experiments, an incision was made on the left side ofthe peritoneal cavity, and the kidney was exposed. A small hole was madein the capsule, and the thymus was inserted between the kidney capsuleand the arenchyma. Light cauterization was used to seal the opening. Thewound was closed with surgical sutures.

Pancreatic Islet Transplantation

Recipient C57BL/6 mice were injected with 190 mg per kg of body weightstreptozotocin (Sigma) intraperitoneally to induce diabetes. Diabetes(blood glucose concentration greater than 300 mg/dl) was confirmed 3 dafter injection using a Contour Blood Glucose Meter (Bayer). Thepancreases from adult C57BL/6 GFP+ transgenic mice were perfused withCollagenase P (Roche) through the bile duct. Digested islets were washedin Hanks Balanced Salt Solution and fetal bovine serum (HBSS/FBS) andplaced in a 70-μm cell strainer. The contents collected in the cellstrainer were transferred to a Petri dish, and individual islets werepicked and counted under a dissecting microscope. Approximately 200-300islets were mixed with 10 μl of Matrigel and loaded into a 27 G insulinsyringe on ice. For lymph node transplantation, a small incision wasmade in the abdomen to expose the jejunal lymph node. The syringecontaining the islets was inserted into the lymph node, and the isletswere slowly injected. For kidney capsule transplantation, a smallincision over the left kidney was made to expose the kidney. A hole wasmade in the capsule, and the islets were delivered in a manner similarto that used for the lymph node. Light cauterization was used to sealthe opening.

The wound was then closed with surgical sutures. If a decrease inglucose concentration was not observed after 1 week, a second islettransplant was performed in the same lymph node.

In Vivo Imaging

To detect donor luciferase-positive C57BL/6 hepatocytes, we used anIVIS200 system (Caliper LifeScience) after intraperitoneal injection of200 μl of 30 mg/ml luciferin substrate into recipient C57BL/6 mice andanesthesia with 3% isoflurane. Images were analyzed using LivingImagesoftware (Caliper LifeScience).

Proliferation Assay

Primary hepatocytes from C57BL/6 GFP+ mice were transplanted into 11mice, as described above. Recipient mice were given drinking watercontaining 0.8 mg/ml BrdU immediately after surgery. After 1 week, weeuthanized three mice for analysis. Partial hepatectomy (PHx), in whichtwo-thirds of the liver of a mouse is removed, was then performed on 5of the 11 mice, and the remaining 3 mice were used for controls. Oneweek later, all eight mice were euthanized for analysis. Usinghistological sections, we determined the amount of hepatocyteengraftment in the lymph nodes by counting GFP+ hepatocytes anddetermined the ratio of proliferating hepatocytes by counting BrdU+hepatocytes.

Allogeneic Transplantation

Primary hepatocytes from C57BL/6 GFP+ mice were transplanted into ten129sv Fah−/− mice by lymph node or splenic injection. Five out of tenmice from each group were intraperitoneally injected with theimmunosuppressive drugs CTLA4-Ig (0.25 mg) and MR1 (0.25 mg), a kindgift from F. Lakkis (University of Pittsburgh), on days 0, 2, 4 and 6after transplantation.

Antibodies

Antibodies specific to the following antigens were purchased forimmunohistochemistry: ER-TR7, LYVE1, GFP, glutamine synthetase, CCR7,S1PR1 (EDG1), F4/80 (Abcam), PNAd, B220, CD4, CD8, CD31, CD105 and Gr-1(BD Biosciences), BrdU (Santa Cruz Biotechnology), DPPIV (AbD Serotec),E-cadherin (Zymed), Collagen IV (SouthernBiotech), keratin 5 (Covance),keratin 8 (DSHB) and C-peptide and glucagon (Cell SignalingTechnologies). Antibodies specific to the following antigens werepurchased for flow cytometric analysis: APC mouse CD3-ε, APC-Cy7 mouseCD8-α, phycoerythrin (PE)-Cy7 mouse CD45 and PE mouse CD4, a mouse VβTCR screening panel, a mouse naive and memory T cell panel (Pharmingen)and a mouse regulatory T (Treg) cell detection kit (Miltenyi).Appropriate isotype control antibodies (BD Biosciences) were used toestimate background fluorescence.

Immunohistochemistry

Tissue was fixed in 4% paraformaldehyde for 4 h, stored in 30% sucrosefor 12 h and then embedded in optimal cutting temperature (OCT) medium,frozen and stored at −80° C. Sections 5-10 (5-10 (m) were mounted onglass slides and fixed in cold acetone for 10 min. Forimmunohistochemical staining, sections were washed with PBS and blockedwith 5% bovine serum albumin (BSA) or milk for 30 min. Sections werethen incubated with primary antibody for 1 h and secondary antibody for30 min. Sections were mounted with Hoechst mounting media. Images werecaptured with an Olympus FluoView 1000 Confocal Microscope or an OlympusIX71 inverted microscope.

Flow Cytometry

Whole blood was collected in K2EDTA collection tubes (Terumo Medical).One hundred microliters of blood was added to coldfluorescence-activated cell sorting (FACS) tubes. Antibodies were addedat a dilution of 1/10 in blood and mixed by gentle pipetting. Reactionswere incubated in the dark in an ice slurry bath for 30 min. Threemilliliters of Red Blood Cell Lysing Buffer (Sigma) was added to eachtube, lightly vortexed and incubated for an additional 5 min. Twomilliliters of flow buffer (2% FBS in HBSS) was added to the tubes,mixed and centrifuged at 500 g for 5 min. The supernatant was aspirated,and secondary antibody was added at a dilution of 1/50 in flow bufferand mixed by gentle pipetting. Reactions were incubated in the dark inan ice slurry bath for 15 min. The red blood cell lysis and centrifugewere repeated as described. The final cell pellet was resuspended in 400μl of flow buffer with propidium iodide. Cells were analyzed using theBD FACSVantage Cell Sorter, a BD Aria II Cell Sorter or a MiltenyiMACSQuant.

Tail Skin Graft

Recipient mice (BALB/c nude) containing an ectopic thymus (C57BL/6) inthe lymph node were anesthetized, and the tail skin (5 mm×5 mm). fromCBA/CaJ or C57BL/6 mice was grafted on the left and right lateral sidesof the superior dorsal region of the recipient mouse, respectively. Abandage was applied and removed 7 d after surgery. The grafts wereobserved daily for rejection.

Tumor Cell Transplantation

Human metastatic colon cancer cells (Tu #12) were prepared andtransplanted into mice as previously described (32). Briefly, 3×10⁵cells were transplanted by subcutaneous injections with Matrigel. Tumorsize was recorded once per week using a caliper and calculated as(π/6)×(length (mm)×width2 (mm²)).

LPS-Induced Inflammation

LPS (1 mg per kg of body weight, Sigma) was injected intraperitoneallyinto C57BL/6 wild-type or normoglycemic mice that previously received anislet transplant to the lymph node. Mice were bled to determine glucoseconcentrations or measure serum cytokine levels. Cytokine level wasdetermined by ELISA assay for TNF-α, IL-1β and IL-6 (eBioscience).

Statistical Analyses

Statistical significance was determined with an unpaired two-tailedStudent's t-test for the data shown in FIGS. 1E, 2D and 3A.

Results Direct Injection of Hepatocytes into the Lymph Node

We targeted single jejunal lymph nodes in the abdominal cavities ofC57BL/6 wild-type mice, an area that we selected because it is easilyaccessible and relatively large (13). After injection of 100,000-500,000syngeneic primary hepatocytes, the lymph nodes enlarged slightly and hadno visible leakage, as evidenced by staining with Evans blue dye (FIG.1A). One day after injection, syngeneic luciferase-expressinghepatocytes remained in the lymph nodes, as determined by in vivoimaging of luciferase (FIG. 1B). In contrast, splenic andintraperitoneal injection of hepatocytes resulted in the rapiddispersion of cells to the liver and throughout the abdominal cavity,respectively (FIG. 1B). One week after transplant, syngeneic GFP+hepatocytes were distributed mainly in the subcapsular sinus of thelymph nodes but were not present in the lymph node follicles or germinalcenters (FIG. 1C). Additionally, the hepatocytes rapidly formed patchesof hepatic tissue expressing E-cadherin (FIG. 1C). Thishepatocyte-to-hepatocyte attachment may help retain hepatocytes withinthe site of transplantation (14).

Furthermore, the cell trafficking molecule sphingosine-1-phosphatereceptor 1 (S1PR1), which is necessary for the egress of B and T cellsfrom lymph nodes (15,16), was not present on hepatocytes in the lymphnodes. Conversely, CCR7, a molecule known to control the migration ofmemory T cells and/or tumor cells into the lymph nodes (17), wasexpressed by hepatocytes in the lymph nodes (FIG. 1D). Theseobservations suggest a potential mechanism of retention of healthyhepatocytes in the lymph node.

To assess whether the engrafted hepatocytes in the lymph nodesproliferate in response to growth stimuli in C57BL/6 wild-type recipientmice, we performed partial hepatectomy (PHx) 1 week after syngeneichepatocyte injection into the lymph nodes and added bromodeoxyuridine(BrdU) to the drinking water of the recipient mice. PHx is aphysiologically relevant method of stimulating hepatocyte growth 18. Weanalyzed BrdU incorporation in the lymph node-engrafted hepatocytes 2weeks after injection and compared the result to that in mice that werenot subjected to PHx. The number of GFP-expressing hepatocytes and thepercentage of BrdU+ hepatocytes in the lymph nodes of the mice subjectedto PHx were significantly higher than those in the mice not subjected toPHx (FIG. 1E), indicating that hepatocytes in the lymph nodes respond togrowth stimuli after PHx.

A Functional Ectopic Liver in the Lymph Node

Next, we asked whether transplantation of syngeneic hepatocytes intosingle jejunal lymph nodes of Fah−/− tyrosinemic mice (19-21), a modelof lethal metabolic liver failure, would induce hepatocyteproliferation. Twelve weeks after hepatocyte injection into the lymphnodes, the mice were rescued from lethal liver failure by the newlyhepatized growth in the jejunal lymph nodes (FIG. 2). Hepatocyteengraftment was localized to the transplanted lymph nodes and was notpresent in other lymph nodes or the native liver (FIG. 2A), suggestingthat a lymphatic distribution of hepatocytes did not occur. The‘hepatized’ lymph nodes were composed mostly of newly formed livertissue containing Fah+GFP+ hepatocytes but also included remnants of thelymphatic system, as revealed by surrounding LYVE1+ lymphaticendothelial cells (FIG. 2B). The lymph nodes were transformed intohepatic organoids composed of characteristically cuboidal hepatocytesbut with the absence of a biliary system (12). To define the hepatizedlymph node microarchitecture, we stained recipient lymph nodes forER-TR7, a marker of fibroblastic reticular cells (FRCs), dipeptidylpeptidase-4 (DPP IV), a marker for brush borders of hepatocytes and bilecanaliculi organization, and glutamine synthetase, a marker of zonalityfor hepatocytes surrounding the terminal hepatic venules 22 (FIG. 2B).GFP+ hepatocytes resided in cellular proximity with ER-TR7+ FRCs. FRCsare known to have a crucial role in establishing the reticular network,as well as in regulating immune function (11,23). Similar to what isobserved in normal liver anatomy, DPP IV was localized throughout thehepatized lymph nodes. Moreover, we identified glutaminesynthetase-positive hepatocytes surrounding some of the hepatic veins inthe newly formed liver tissues. Notably, we observed survival of themice (FIG. 2C) and long-term persistence (over 25 weeks) of the graftafter transplantation to the lymph nodes (FIG. 2A), and we found noimmune responsiveness to the grafts, as indicated by the presence ofvery few lymphocytes and macrophages around the transplantation zones(FIG. 6).

We then tested whether this transplantation method is effective in otherlymph nodes throughout the body, as all lymph nodes share a commonhistological architecture (9,13,24). We injected syngeneic hepatocytesinto single extra-abdominal axillary or popliteal lymph nodes in Fah−/−mice (FIG. 2A). Similar to the results from the intra-abdominal jejunallymph node injection, we observed a single, large, hepatized lymph nodeseveral weeks after cell transplantation, which subsequently rescuedmice from lethal liver failure (FIG. 2C). In wild-type mice, a jejunallymph node weighs around 5±3 mg (mean±s.d.), whereas a popliteal oraxillary lymph node weighs 3±1 mg (n=5). Because the jejunal lymph nodeis the largest lymph node in the mouse, it was easier to inject, andinjection at this site led to a higher survival rate (FIG. 2C).Furthermore, when we compared the mass of hepatic tissue generated inthe intra-abdominal jejunal lymph nodes to those generated in theextra-abdominal axillary and popliteal lymph nodes, we found nostatistical differences, indicating no tissue growth advantage betweenthese three lymph nodes (FIG. 2D). Transplantation of hepatocytes intoperiportal lymph nodes, one of the closest lymph nodes to the liver,also resulted in large hepatized lymph nodes and rescued the recipientmice from lethal liver disease. However, transplantation of lymph nodescloser to the liver did not seem to be beneficial for the experimentaloutcome when compared with the other transplanted lymph nodes (FIG. 2D).

Thus far, all transplantations used syngeneic donor and recipient mice.Because lymph nodes perform a central function for allogeneicrecognition (25), we next tested whether successful engraftment ispossible in allogeneic lymph nodes. We injected C57BL/6 GFP+ hepatocytesinto the liver (through splenic injection) or into the lymph nodes of129sv Fah−/− recipient mice. 129sv and C57BL/6 mice share the majorhistocompatibility complex haplotype H2b but differ in their minorhistocompatibility antigens and are considered allogeneic (26,27).Whereas C57BL/6 hepatocytes transplanted into 129sv Fah−/− recipientsdid not engraft (FIG. 2E), blocking the co-stimulation pathways CD28-B7(blocked by CTLA4-Ig) and CD40−CD40L (blocked by MR1)(28,29) in the129sv Fah−/− recipients facilitated successful engraftment of C57BL/6GFP+ hepatocytes injected into either the spleen or the lymph node (FIG.2E). This result shows that the immune reaction to transplanted cells inthe lymph nodes is not stronger or faster compared to the reaction tocells transplanted in another site.

A functional Ectopic Thymus in the Lymph Node

Using a similar approach, we asked whether de novo thymus function couldbe generated in the lymph nodes of athymic mice. We harvested thymusesfrom newborn C57BL/6 GFP+ transgenic mice and minced and injected themdirectly into the jejunal lymph nodes or under the kidney capsules ofathymic BALB/c nude mice. After 1 month, we analyzed the blood of therecipient mice by flow cytometry for the presence of recipient (GFP−)single-positive CD4+ and CD8+ T cells. Thymic transplant into the lymphnode (LN-Tx nude mice) or under the kidney capsule (KC-Tx nude mice)generated circulating recipient single-positive CD4+ and CD8+ T cells(FIG. 3A and FIG. 7). Ten months after transplantation, single-positiveCD4+ and CD8+ T cells were still present in the peripheral blood,indicating long-term thymic engraftment in the lymph nodes (FIG. 8).Notably, transplantation of a thymic single-cell suspension alsoresulted in the presence of single-positive CD4+ and CD8+ T cells,although to a lesser degree than did transplantation of minced thymictissues (Table 1=FIG. 9). We used newborn GFP+ mice as a source of donorthymuses because they contain a minimal amount of mature thymocytes. Infact, less than 6% (n=3/54) of the recipient mice showed any donor GFP+T-cell contamination in the blood 1 month after transplant when measuredby flow cytometry. We excluded these three mice from further studies.

We then harvested the ectopic thymuses for further characterization ofthe engraftment. GFP+ epithelial thymic cells remained within theinjected lymph nodes and were organized into an epithelial thymicstructure (FIG. 3B). Previously, thymic epithelia have beendistinguished by their cytokeratin 5 (K5) and cytokeratin 8 (K8)phenotypes (30). The ectopic thymuses were present in the subcapsularsinus of the lymph nodes (FIG. 10) and contained both K5- andK8-positive regions, which correspond with thymic medullary and corticalepithelia, respectively (FIG. 3B). We also analyzed the ectopic thymusesfor the presence of recipient double-positive CD4+ CD8+ thymocytes,which represent immature T cells undergoing thymic selection (31). Theectopic thymuses contained recipient double-positive thymocytes as wellas single-positive CD4+ and CD8+ T cells, indicating a selectivemechanism of T-cell commitment and maturation (FIG. 3B).

To more fully characterize the T-cell phenotypes generated in the LN-Txnude mice, we analyzed the T-cell receptor (TCR) repertoire by flowcytometry staining with antibodies recognizing different TCR Vβ segments(Vβ 2, Vβ 3, Vβ 4, Vβ 5.1,5.2, Vβ 6, Vβ 7, Vβ 8.1,8.2, Vβ 8.3, Vβ 9, VβP 10b, Vβ 11, Vβ 12, Vβ 13, Vβ 14 and Vβ 17a). We found each V Vβsegment on the CD3+ T cells of C57BL/6 wild-type splenocytes.Heterozygous BALB/c nude mouse splenocytes expressed 10 out of the 15 Vβsegments, which is consistent with known partial or complete geneticdeletions of certain Vβ segments (Vβ 3, Vβ 5.1,5.2, Vβ 9, Vβ 11 and Vβ12) in this strain. Notably, splenocytes in LN-Tx nude recipientsexpressed a Vβ profile similar to that of the heterozygous BALB/c nudemice (FIG. 3C). These results demonstrate that a C57BL/6 ectopic thymuspromotes the development of T cells generated from the bone marrow ofrecipient BALB/c nude mice. We then further characterized the splenic Tcells in LN-Tx nude mice by flow cytometry to determine whetherregulatory, naive, central memory and effector memory T cell subsetswere present. We detected regulatory T cells by analyzing FoxP3expression in CD4+CD25+ T cells. We observed similar percentages ofregulatory T cells in splenocytes from C57BL/6, heterozygous nude andLN-Tx nude mice (FIG. 3D). We distinguished naive and memory T cellsusing differential expression of CD44 and CD62L (where naive cells areCD44−CD62L+, central memory cells are CD44+CD62L+, and effector memorycells are CD44+CD62L−). As with the regulatory T cells, we detectednaive and memory T cell subsets among splenocytes from C57BL/6,heterozygous nude and LN-Tx nude mice (FIG. 3D). The percentage ofeffector memory T cells was higher and the percentage of naive T cellswas lower in the LN-Tx nude mice than in the other two groups, perhapsbecause these mice had been previously exposed to skin grafts (seebelow).

As the LN-Tx nude mice contained a range of peripheral single-positiveCD4+ and CD8+ T cells, we asked whether the de novo immune system inthese recipient mice could mount a T cell-mediated response against askin allograft. We transplanted tail skin from C57BL/6 (syngeneic withregard to the donor thymus) or CBA/CaJ (allogeneic with regard to thedonor thymus) mice to the dorsal side of LN-Tx BALB/c nude mice. After 2weeks, all of the LN-Tx BALB/c nude mice had rejected the allogeneicCBA/CaJ skin grafts. Conversely, the C57BL/6 skin grafts were allcompletely accepted after 2 weeks (FIG. 3E). We then asked whether LN-TxBALB/c nude mice would mount an immune response against a xenogeneictumor cell transplant. We thus injected 300,000 human colorectal cancercells (32) into the subcutaneous space of BALB/c nude and LN-Tx BALB/cnude mice. The majority (8 of 9) of the injected LN-Tx BALB/c nude micerejected the tumors (FIG. 3E). In contrast, xenogeneic tumors grew inthe untreated BALB/c nude mice (FIG. 3E). These results suggest that thesingle-positive T cells present in the LN-Tx BALB/c nude mice werefunctional. Together, these data support the concept of using the lymphnode as a site for thymic transplant to generate an ectopic thymus.

Functional Pancreatic Islets in the Lymph Node

We then hypothesized that the lymph node might also provide a suitableenvironment for pancreatic islet transplantation. We harvested isletsfrom C57BL/6 GFP+ transgenic mice and transplanted them into the jejunallymph nodes of C57BL/6 wild-type mice (LN-Tx mice) treated withstreptozotocin, which induces diabetes. We monitored the blood glucoseconcentrations of these mice weekly, and after 6 weeks we removed thetransplanted lymph nodes to analyze islet engraftment. We found GFP+islets within the subcapsular sinus of the lymph nodes adjacent to thedensely packed lymphocytes that are indicative of a typical lymph node(FIG. 4A). We detected expression of C-peptide and glucagon, markers ofpancreatic β-cell and α-cell function, respectively, within theengrafted lymph nodes (FIG. 4A).

Next, we asked whether islets transplanted to the lymph nodes ofdiabetic mice (blood glucose concentrations greater than 300 mg/dl) areable to lower the blood glucose concentrations of the mice to a normalrange (100-200 mg/dl). Similarly to islets transplanted under the kidneycapsule, islets transplanted directly into the lymph nodes restored themean blood glucose concentrations of the recipient mice to normal levelswithin 6 weeks after transplantation (FIG. 4B). In three of five LN-Txmice, a second islet transplant was performed into the jejunal lymphnode 1 week after the initial transplant. The mice that did not receivea transplant all died after 3 weeks. Furthermore, normoglycemia wasmaintained in the one mouse examined at 6 months for at least 6 monthsafter lymph node transplantation. These results suggest that pancreaticislets transplanted in the lymph nodes survive and function in vivo withthe capacity to sustain long-term normoglycemia.

Because we observed engraftment of multiple cell types in the lymphnodes, we asked whether activation of a lymph node-mediated immuneresponse might interfere with the function of the engrafted cells.Therefore, we induced an inflammatory reaction in the intraperitonealcavities of normoglycemic mice that had received islet transplants intotheir lymph nodes; to induce the inflammatory reaction, we injected themice with lipopolysaccharide (LPS, 1 mg per kg of body weight). Weconfirmed the effective induction of inflammation by measuring the serumconcentrations of tumor necrosis factor α (TNF-α), interleukin-1β(IL-1β) and IL-6 (FIG. 4C). Immediately after LPS injection, we noted atemporary reduction in the weight and blood glucose concentrations ofthe mice, which completely normalized after 4 d (FIG. 4C). We did notobserve any increase in glucose concentrations above normal levels inthese mice (normoglycemia was still detected 5 weeks after LPSinjection).

Together, these data suggest no apparent negative effect of thelymphatic or immune systems on lymph node-grafted islets.

Vasculature of Injected Lymph Nodes

We demonstrated that cell engraftment in the lymph node is notrestricted to one cell type. This result is consistent with the currentconcept of metastasis to the lymph node, where multiple cancer celltypes are capable of residing, and suggests that the lymph nodeenvironment is exceptionally well suited to promote the survival andfunction of transplanted cells in general. Because abundant vasculatureis required to sustain organ function (6,33), we hypothesize that thedense vascular network in the lymph node is an important contributor tosustaining long-term engraftment of normal epithelial tissue. Weobserved dense neovascular trees in the lymph nodes into which weinjected hepatocytes (FIG. 5A). Lymph nodes injected with hepatocytes,thymic cells or islets contained many recipient-derived CD31+endothelial cells pervading the areas of engraftment, suggesting thatextensive blood-vessel remodeling took place during the ectopic tissueengraftment (FIG. 5B and Supplementary FIG. 5). Moreover, we detectedCD105+ (Endoglin) cells and Collagen IV+ cells, which are markers ofneovascular remodeling, in each of the engrafted lymph nodes (FIG. 5B)34-36. These data suggest that blood vessels in the surrounding lymphnode environment contribute to the neovascularization and overallfunction of the ectopic tissues.

Discussion

There are over 500 lymph nodes in the human body, many of which arerelatively easily accessible. Although a single lymph node structurallylimits the number of donor cells that can be transplanted, it istechnically feasible to transplant more than one lymph node to gainsufficient organ or tissue function from the transplanted cells. Thepotential loss of function in a few lymph nodes does not seem tocompromise the overall function of the lymphatic system. In fact,lymphedema is the most common complication after lymphadenectomy inpatients with cancer and only affects a limited number of these patients(37). Compression syndrome and lymphatic spread are two of the commonissues in patients with cancer that have metastatic diseases in theirlymph nodes. However, we found none of these complications in any of ourexperiments. It is also important to note that we transplanted healthy,not transformed, cells into the lymph nodes, so the intrinsic potentialof the transformed cells for widespread metastatic migration anduncontrolled growth was not present in our experiments. Large animalstudies will provide further insight into the potential difficultiesassociated with cell transplant to the lymph node.

Lymph node biopsies by fine-needle aspiration are a routine diagnosticprocedure, with lymph nodes often being readily identified by palpationor ultrasound guidance. In fact, the clinical application of lymph nodeinjection has been validated, with patients rating the procedure as lesspainful than venous puncture (38). Moreover, if a less superficial nodeis advantageous for therapy, ultrasound guidance can be used tosuccessfully inject the visceral mesenteric lymph nodes (39). The strongclinical precedent of ultrasound-guided lymph node injections may helpmake this technique readily adaptable to a clinical setting. Theseminimally invasive techniques may also provide a potential therapy forpatients who are ineligible for a more invasive therapy because ofcomorbidities.

In the treatment of liver failure, transplantation of hepatocytes intoectopic sites, including the spleen, pancreas, peritoneal cavity andsubrenal capsule, has been proposed. The feasibility and efficacy ofthese techniques have been confirmed in preclinical studies, butclinical success rates have been limited thus far, and new methods areneeded to improve hepatocyte engraftment (1,7). It should be noted thatour goal is not to replace a whole liver but to complement liverfunctions by generating functional ectopic hepatic tissue. In our Fah−/−mice, we generated around 70% of the liver mass in one lymph node. Thenative liver is still present but at a reduced size and, probably,reduced function. For patients with liver disease, we postulate thatliver function gradually deteriorates and transplantation of severallymph nodes with hepatocytes will create enough hepatic mass tostabilize the liver disease. In addition, we hypothesize that thehepatic mass generated in the lymph node may provide enough hepaticfunction to facilitate regeneration of the native liver. It should alsobe noted that transplantation of heterotopic liver has been discussed atlength in the literature as a possible alternative to orthotopic livertransplantation (34,40,41).

Thymus transplants have been performed exclusively in the quadriceps ofpediatric patients with complete DiGeorge syndrome (4). Unfortunately,children with DiGeorge syndrome often show poor growth, andtransplantation is frequently postponed to allow for further development(42). Furthermore, a lack of vascularization and the resulting ischemiaafter transplant are detrimental (43). Transplanting thymic cells intothe lymph node may represent an advantageous site to provide thymicfunction.

The optimal implantation site for pancreatic islet transplantation toincrease function, reduce necessary implantation mass and decreaseimmunogenicity is still under debate (6,44-46). However, proximity to agood vascular supply is clearly essential for the survival of isletcells, as well as for that of hepatocytes and thymic epithelial cells.

One concern for cell transplant into the lymph node is the rapid immuneresponse that can be initiated by the introduction of a foreign antigeninto a site that is densely packed with lymphocytes. In our study, weincluded experiments under allogeneic conditions and blocked the T-cellresponse to prevent any alloreactivity. We observed that the immunereaction in the lymph nodes is not stronger or faster than the reactionin other sites and that immune suppression therapy works at a similarefficiency compared to the classic hepatocyte transplantation in thespleen. We expect that immunosuppressive therapies similar to those usedin clinical organ transplants can be used with this approach.

Reprogramming somatic cells provides an exciting potential source ofdonor cells for regenerative medicine (47). As these cells can bederived from autologous material and are capable of being recognized as‘self’ by the host immune system, they can potentially overcomeimmunologic barriers. However, recent studies have suggested that theseautologous cells may not be entirely protected from the immune system(48). On the basis of our results, the lymph node may be an effectivetransplantation site for reprogrammed somatic cells that can bedeveloped for organ regeneration purposes.

In summary, we provide the first report, to our knowledge, describingthe use of a lymph node as a site for functional cellular transplant. Bydirectly injecting the lymph node with hepatocytes, thymuses orpancreatic islets, we demonstrate engraftment of the donor cells andsubsequent organ function. This new approach of using the lymph node asan in vivo bioreactor in which to regenerate functional organs may bebeneficial to the field of regenerative medicine.

EXAMPLE 2: MULTIPLICATION OF VARIOUS TISSUES IN THE MOUSE LYMPH NODE

The severe shortage of deceased-donor organs has driven a search foralternative methods of treating patients with failing organs. Cell-basedregenerative medicine is emerging as a promising interdisciplinary fieldfor tissue repair and restoration of organ function, able to contributeto improving health in a minimally invasive fashion. However, cellulartransplantation has limitations including recapitulating the functionsof structurally complex organs. One potential approach to replacing suchfunctions is through organogenesis. Growing new organs in situ can beachieved by transplanting embryonic tissues. Renal capsule grafting is awell established method of growing embryonic or neonatal organ rudimentsin vivo for extended periods. However, space limitation beneath therenal capsule has proven to be an impediment to the growth oftransplants, the limitation increasing with age. Recently, the mousejejunal lymph nodes have been identified as alternative ectopic sitesable to provide a permissive environment for liver, pancreas and thymuscells. We aimed at investigating their capability in also supporting thematuration of multiple other tissues explanted from E14.5-15.5 GFP+C57BL/6 mouse embryos (FIG. 11). Our preliminary data (FIG. 12A-12D)suggests that jejunal lymph nodes can favor the engraftment/maturationof several tissues. Differentiation of lung tissue from apseudoglandular to a mixed saccular/alveolar morphology was observed(FIG. 12B). Crypt-like structures developed following injection ofintestinal fragments into lymph nodes (FIG. 12C). Importantly,goblet-like cells were observed all along these structures, suggestingthe presence of terminally differentiated intestinal cell types.Similarly, well-developed renal corpuscles were found in repopulatedlymph nodes (12D). Further investigations are needed to understandwhether these lymph nodes carry out specific functions. Our lab haspreviously shown that primary hepatocytes injected intraperitoneallyinto mice lacking fumarylacetoacetate hydrolase (a mouse model oftyrosinemia type I) migrate and colonize the host abdominal lymphaticsand restore hepatic functions (1). More recently, hepatocytes andpancreatic islets injected directly into a single lymph node were shownto generate functional tissues, rescuing mice from lethal liver failure,and streptozotocin-induced diabetes, respectively (2). Similarly, denovo thymus function could be generated in athymic mice by injectingthymic tissues into lymph nodes (2). These three independent resultssupport the proof of concept that the lymph node provides a hospitableenvironment for normal cell engraftment. We extended this approach toadditional tissues, demonstrating lymph node capacity in supporting notonly the engraftment, but also the maturation of murine fetal tissues,including lung, intestine and kidney.

Our preliminary results using fetal kidney are particularly encouraging.Morphogenesis of the S-shaped body to a structure that contains vascularloops of the glomerulus and the Bowman's capsule was observed.Engineered glomeruli contained all the different cell types present inthe adult kidney. Whether the implanted tissue generates cells endowedwith the ability to produce erytropoietin and exhibits physiologicfunctions including blood filtration and tubular reabsorption ofmacromolecules, will be investigated in the future.

Beyond its capacity in supporting the maturation of fetal tissues, thelymph node also provided a suitable environment for adult thyroid glandregeneration (FIG. 13). Indeed, follicle-like structures were observedin the repopulated lymph node, suggesting that the thyroid functionmight be restored in human patients after total thyroidectomy, by simplyusing their lymph nodes as bioreactors, thus avoiding thyroid hormonereplacement medication for the rest of their life.

EXAMPLE 3: THE MOUSE LYMPH NODE AS AN EXTOPIC TRANSPLANTATION SITE FORMULTIPLE TISSUES

Fetal tissues were isolated for implantation as set forth in FIG. 11.There are a number of problems associated with determining gestationalage in embryonic mice (see FIGS. 14A-14C). Injections of various fetaltissues were performed and in many cases resulted in lymph noderepopulation (FIG. 15). FIG. 16 shows exemplary transplantation ofthyroid gland tissue into lymph node. Transplantation of liver tissue inlymph node is shown in FIG. 17. FIG. 18 shows the results oftransplanting brain tissue into lymph node; brain tissue was observed togrow well in lymph node. As shown in FIG. 19, transplantation of lungtissue into lymph node was observed to result in differentiation of lungtissue from a pseudoglandular to a mixed saccular/alveolar morphology.FIG. 20 shows that when intestinal tissue was transplanted into lymphnode, crypt-like structures developed, and goblet-like cells wereobserved along the crypt-like structures, suggesting the presence ofterminally-differentiated intestinal cell types. Of note, due to itssize, the lymph node would not be appropriate for organogenesis. FIGS.21A-21C show the results of kidney tissue transplant into lymph nodeshowing the presence of renal-like histology and the presence of renalcell markers. In particular, a well-developed renal corpuscle was foundwithin GFP+ tissue inside the repopulated lymph node, and its meanvolume was increased 3-fold with respect to parental tissue.Interestingly, the glomerulus-like structure was not GFP+. GK2, GK3, andGK4 lymph nodes were injected with kidneys isolated from embryos derivedfrom the same mother, however, well-developed renal corpuscles wereobserved only in the GK3 lymph node. This could reflect variabilityamong embryos or recipient mice. Further results relating to thetransplant of kidney tissue into lymph node are shown in FIGS. 22A-22D.These results, inter alia, indicate that the mouse lymph node cansupport the maturation of kidney. Morphogenesis of the S-shaped body toa structure that contains vascular loops of the glomerulus and Bowman'scapsule was observed.

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EXAMPLE 4: THE MOUSE LYMPH NODE AS AN ECTOPIC NICHE FOR KIDNEYORGANOGENESIS Summary

The shortage of organs for kidney transplantation has created the needfor new strategies to regenerate renal functions. Here we provide thefirst evidence that the lymph node permitted mouse metanephroi toengraft, mature and perform glomerular filtration. Host cells likelycontributed to this process. Over time, production of waste fluidresulted in some cases in graft degeneration. Indeed, urine-likefluid-containing cysts and glomerular alterations were observed inseveral grafts after 12 weeks post transplantation. Importantly, thekidney graft adapted in response to a loss of host renal mass, speedingits development. Thus, the lymph node provides a unique tool forstudying the mechanisms of renal maturation or cell proliferation andfluid secretion. This innovative system can also be used to validate thedifferentiation potential of candidate cells in regenerative nephrologyand may provide an exclusive site for kidney organogenesis andregeneration.

Methods Tissue Collection and Transplantation

E14.5/15.5 kidneys were retrieved from timed pregnant GFP+ or wild-typeC57BL/6 black mice under a dissecting microscope (embryos wereconsidered 0.5 days old when the vaginal plug was detected in themorning). Alternatively, kidneys were isolated from 3-day-old (P3) GFP+C57BL/6 black mice. All kidneys were chopped in PBS and kept on iceuntil injection. For lymph node transplantation, recipient mice(6-week-old wild-type C57BL/6 black mice, n=52) were anesthetized with1-3% isoflurane. A small incision was made in the abdomen to exposejejunal lymph nodes. A 1000 μL threaded plunger syringe (Hamilton,81341) with a removable needle (gauge 20) was used to slowly injectkidney fragments into a single lymph node (paired kidneys from an embryoper mouse were injected). Light cauterization was used to seal theopening. The wound was then closed with surgical sutures. Ketoprofentreatment (2 mg/kg, IM) for postoperative pain relief was initiatedright after surgery, and continued for 2 additional consecutive days.After 3, 6, 12 or 16 weeks from transplantation, mice were euthanizedfor analysis. Mice were bred and housed in the Division of LaboratoryAnimal Resources facility at the University of Pittsburgh Center forBiotechnology and Bioengineering. Experimental protocols followed USNational Institutes of Health guidelines for animal care and wereapproved by the Institutional Animal Care and Use Committee at theUniversity of Pittsburgh.

Histology and Immunofluorescence/Immunohistochemistry

Repopulated jejunal lymph nodes and kidneys were fixed 2-4 hours in 4%PFA, and embedded in OCT or paraffin for further analysis. Hematoxylinand eosin (H&E), Periodic acid—Schiff (PAS), Masson's trichrome (TRI),and Picro-sirius red (PSR) stains were performed as described elsewhere.Sections were also stained with antibodies against ER-TR7 (Abcam,ab51824), Collagen IV (SB, 1340-01), CD31 (BD, 550274), Podoplanin(Angiobio, 11- 033), Claudin-2 (Abcam, ab76032), Keratin-8 (DSHB,TROMA-1), Erythropoietin (SCBT, sc-7956), GFP (Abcam, ab6556), BrdU(SCBT, sc-32323), AQP1 (Abcam, ab15080), NKCC2 (SCBT, sc-133823), AQP2(Abcam, ab85876), CD45 (BD, 550539), CD106 (SCBT, sc-8304), CD3 (BD,550275), CD4 (550280), CD8 (BD, 553027), CD45R/B220 (BD, 550286), Ly6C/G(BD, 550291), F4/80 (Caltag Lab, MF48015), WT-1 (SCBT, sc-192) or Epcam(Abcam, ab32392). For BrdU staining, sections were incubated in 2N HClfor 30 minutes in order to denature DNA. Five min incubation in 0.1 Mborate buffer pH 8.0 was then carried out to neutralize the acid.Finally, BrdU antibody was added. Alexa Fluor 594 (Invitrogen) orbiotinylated (Dako, LSAB2 System-HRP) secondary antibodies were used todetect primary antibodies. Biotin labeling was then revealed usingstreptavidin-HRP conjugate (Dako, LSAB2 System-HRP) and AECsubstrate-chromogen (BioGenex, HK129-5K). Nuclei were counterstainedusing Hoechst or hematoxylin.

RNA Extraction, cDNA Synthesis, RT-PCR

Total RNA was isolated from tissues stored in RNAlater® reagent (QIAGEN)using the RNeasy Mini kit (QIAGEN), according to the manufacturer'sinstructions. Potentially contaminating genomic DNA was digested usingDNase (QIAGEN). cDNA was synthesized using the iScript™ ReverseTranscription Supermix for RT-qPCR (Bio-Rad). PCR was performed usingthe iTaq DNA Polymerase kit (Bio-Rad). GAPDH transcript levels served asthe housekeeping control target. Sequences of primers were as follows:UT-A1, Fwd, 5′-GACAGTGAGACGCAGTGAAG-3′ (SEQ ID NO: 1), Rev,5′-ACGGTCTCAGAGCTCTCTTC-3′ (SEQ ID NO: 2); UTA2, Fwd,5′-TTTCTCCAGTCCTATCTGAG-3′ (SEQ ID NO: 3), Rev,5′-ACGGTCTCAGAGCTCTCTTC-3′ (SEQ ID NO: 2); UT-A3, Fwd,CCTGACAGTGAGACGCAGTG-3′ (SEQ ID NO: 4), Rev, 5′-AGAGTGGAGGCCACACGGAT-3′(SEQ ID NO: 5); UT-B, Fwd, 5′-TCTTCTCAAACAAGGGCGAC-3′(SEQ ID NO: 6),Rev, TTGCTGAGCACGGAGCTCAA-3′ (SEQ ID NO: 7); EPO, Fwd,5′-AAACTGAAGCTGTACACGGGAGA-3′ (SEQ ID NO: 8), Rev,5′-GGAGCAAGTTCGTCGGTCC-3′ (SEQ ID NO: 9); GAPDH, Fwd,5′-GGCATCCTGGGCTACACTGA-3′ (SEQ ID NO: 10), Rev,5′-GGAGTGGGTGTCGCTGTTG-3′(SEQ ID NO: 11). To enhance EPO production,anemia was induced by a rapid withdrawal of blood for 3 consecutive daysbefore mouse sacrifice and lymph node collection.

Blood Urea Nitrogen (BUN) Test

BUN test was performed on both serum and lymph node fluid of micefollowing 16 weeks from embryonic kidney injection (n=5 experimentalmice, plus n=1 control mouse). Blood was collected into polyethyleneterephthalate serum-gel-separator tubes (Terumo Medical). Tubes werethen centrifuged at maximum speed for 10 min, and serum collected. Forlymph fluid collection, briefly, the GFP+ area inside each lymph nodewas isolated under a fluorescent microscope, chopped in very smallpieces, centrifuged at maximum speed for 10 min, and supernatantcollected. BUN test was performed using the QuantiChrom Urea Assay Kit(Bioassay Systems, DIUR-500) according to the manufacturer'sinstructions.

Generation of Chimeric Mice

Bone marrow cells were harvested from tibias and femurs of a GFP+C57BL/6 mouse, as described elsewhere. Subsequently, 6-week-oldwild-type C57BL/6 mice (n=11) were lethally irradiated (1000 rad) andimmediately retro-orbitally infused with 106 donor cells. Mice weretreated with Sulfamethoxazole (SMZ) in the drinking water.

Flow Cytometry

Whole blood was collected in K2EDTA collection tubes (Terumo Medical).One hundred microliters of blood was added to coldfluorescence-activated cell sorting (FACS) tubes. Three milliliters ofRed Blood Cell Lysing Buffer (Sigma) was added to each tube, lightlyvortexed and incubated for 15 min. Two milliliters of flow buffer (2%FBS in HBSS) was added to the tubes, mixed and centrifuged at 500 g for5 min. The supernatant was aspirated. The red blood cell lysis andcentrifuge were repeated as described. The final cell pellet wasresuspended in 400 μl of flow buffer with Sytox Blue dye. Cells wereanalyzed for GFP positivity using a Miltenyi MACSQuant and FlowJosoftware (Tree Star).

Antibodies were added at a dilution of 1/50 in blood and mixed by gentlepipetting. Antibodies used were as follows: PerCP Cy5.5 CD45 (BD,550994), APC CD3 (BD, 553066), PE CD4 (BD, 553730), APC Cy7 CD8 (BD,557654), PE CD45R/B220 (BD, 553090), APC CD19 (BD, 550992), PE CD11b(BD, 553311), and APC Ly6G−Ly6C (BD, 553129). Reactions were incubatedin the dark in an ice slurry bath for one hour. Three milliliters of RedBlood Cell Lysing Buffer (Sigma) was added to each tube, lightlyvortexed and incubated for an additional 15 min. Two milliliters of flowbuffer (2% FBS in HBSS) was added to the tubes, mixed and centrifuged at500 g for 5 min. The supernatant was aspirated. The red blood cell lysisand centrifuge were repeated as described. The final cell pellet wasresuspended in 400 μl of flow buffer with Sytox Blue dye. Cells wereanalyzed using a Miltenyi MACSQuant and FlowJo software (Tree Star).

Proliferation Assay

To assess proliferation of 6-week ectopic kidneys 1 mg BrdU wasdissolved in 200 ul of PBS and intraperitoneally injected 24 hoursbefore mouse sacrifice. To assess ectopic kidney proliferation inresponse to growth stimuli, embryonic kidneys from GFP+ C57BL/6 blackmice were transplanted into 9 wild-type C57BL/6 black mice, as describedabove. Following 12 days from transplantation, 5 of the 9 recipient micereceived left nephrectomy (Nx), while the remaining 4 mice received asham operation. All mice were given drinking water containing 0.8 mg/mlBrdU and 1% sucrose immediately after surgery. BrdUcontaining drinkingwater was prepared fresh and replaced daily for 9 consecutive days,after which, it was replaced with regular water. After 5 additionaldays, all mice were euthanized for analysis. The number of BrdU-positiveproliferating cells in the recipient kidneys was determined per renalcross-section.

Statistical Analysis

Data are presented as means±SD. Statistical analysis was performed usingStudent's t test (p<0.05 was considered significant).

Immunofluorescence

Repopulated jejunal lymph nodes were fixed 2 hours in 4% PFA, andembedded in OCT for further analysis. Sections were stained withantibodies against Podoplanin (Angiobio, 11-033), Claudin-2 (Abcam,ab76032), WT-1 (SCBT, sc-192), CD31 (BD, 550274), Keratin-8 (DSHB,TROMA-1) or Vimentin (SCBT, sc-5565). Alexa Fluor 594 antibodies(Invitrogen) were used to detect primary antibodies. Nuclei werecounterstained using Hoechst.

Results The Lymph Node is a Permissive Site for Kidney Organogenesis

We first investigated whether mid-embryonic mouse kidney fragments couldbecome integrated into a host mouse lymph node, and undergomorphological maturation. Renal tissues were harvested from C57BL/6 GFP+transgenic embryos, isolated from ureteric buds, minced, and injecteddirectly into a single jejunal lymph node of adult wild-type C57BL/6mice (FIG. 23A). Following 3 weeks, recipient mice were sacrificed,lymph nodes collected, and histologically examined. Morphogenesis ofS-shaped bodies into more mature renal corpuscles was observed inectopic grafts 3 weeks after embryonic kidney transplantation (FIG.23B). Developing renal corpuscles expressed type IV collagen in theirglomerular basement membranes (GBM) as well in mesangial areas (FIG.23C). Three stages of glomerulus maturation could be distinguished basedon the literature (FIG. 23C) [18]. Briefly, glomeruli with loosestructure of the tuft, and capillary loops lined with typical flatepithelia were defined as mature glomeruli. Glomeruli with less thanhalf the circumference of capillary loops lined with cuboidal epithelialcells, but at least five of them adjoining, were defined as intermediateglomeruli. Finally, glomeruli with at least half of the circumference ofcapillary loops densely lined with cuboidal epithelial cells weredefined as immature glomeruli. Ectopic grafts also showed some S-shapedbodies, further indicating that kidney maturation was not completed atthe time of lymph node collection. Nevertheless, mature ectopicglomeruli contained different cell types present in the adultglomerulus, including CD31-positive endothelial cells, andpodoplaninpositive podocytes (FIG. 23D). Developing kidneys alsocomprised rudimental claudin-2-and keratin-8-positive tubules (FIG.23D). Importantly, ectopic grafts showed tubular erythropoietin (Epo)expression, indicating hormonal competence (FIG. 23D).

We also found that kidney organogenesis into the lymph node wascritically dependent on the stage of renal development at the time oftransplantation. Despite 3-day-old mice (P3) kidneys show glomerularmaturity, they failed to efficiently engraft into the lymph node (FIG.29). Embryonic day (E) 14.5 to 15.5 kidneys generated larger and thickergrafts as compared to P3 kidneys following 3 weeks from transplantationinto the lymph node. Moreover, while embryonic kidneys acquired moremature morphological characteristics into the lymph node, newbornkidneys failed to recapitulate their native morphology, resulting in animperfect glomerulogenesis. Even extending the growth of newborn kidneyfragments into the lymph node up to 12 weeks did not result in a betterengraftment and maturation, confirming the idea that fetal kidneyharbors more regenerative potential than newborn kidney.

Ectopic Grafts Show Well-Defined, and Proliferating Nephrons withUrineconcentrating Ability 6 Weeks after Transplantation

Three weeks after transplantation, it was not possible to confirm thepresence of mature, functional nephrons. However, some mature nephronswere distinguishable 6 weeks after transplantation (FIG. 24A). Indeed,attached to the developed renal corpuscles, various segments of therenal tubule could be observed. Furthermore, the presence oferythrocytes inside the glomerulus capillary tuft of these elongatedstructures indicated a probable blood filtration capacity (FIG. 24A).Importantly, such structures were still proliferating at the time oflymph node collection, as indicated by bromodeoxyuridine (BrdU)incorporation, administered to the mouse 24 hours before its sacrifice(FIG. 24B). Importantly, 6-week ectopic grafts showedurine-concentrating ability, as indicated by RT-PCR analysis ofdifferent urea transporters (UT-A1, UT-A2, UT-A3, and UT-B) (FIG. 24C).It is not surprising that UT-B mRNA was detected in both control andrepopulated lymph nodes, since this urea transporter is known to beexpressed in non-renal tissues, as well as in erythrocytes [19].Erythropoietin production was also confirmed in 6-week ectopic grafts byRT-PCR analysis of mRNA isolated from phlebotomized mice.

Host Cells Vascularize Kidney Ectopic Graft and Likely Integrate intothe Developing Tissue

In some mice, all nephrons were mature by 12 weeks. These nephronsshowed glomerular expression of podoplanin and CD31 (FIG. 25, left).CD31 staining also indicated that ectopic nephrons were vascularized byhost arterioles (FIG. 25, right). Collagen IV was localized at GBM andtubules (FIG. 25, left), as well in the mesangial areas in the glomeruli(FIG. 25, right), and it likely had a hybrid origin. Indeed, it did notalways colocalize with GFP+ cells. Ectopic nephrons also showedkeratin-8- and erythropoietin-positive tubules (FIG. 25, left).

Renal Cysts Develop within Repopulated Lymph Nodes as a Result ofEctopic Kidney's Ability to Filter the Blood and Produce Urine

While in some mice the ectopic kidney graft was viable and apparentlyfunctional at 12 weeks, in other mice, at the same time point, itcomprised fluid-filled cysts. Renal cystic disease has multipleetiologies. Renal cysts can result from defective differentiation ofkidney tubules [20]. However, while proliferative activity of the renaltubular epithelium is an essential component of cyst formation, fluidsecretion could have a commanding role in cyst development and expansion[21]. We believe in a scenario in which urine-like fluid is produced byectopic kidneys as a result of their functional maturation into thelymph node. In hepatized lymph nodes, ectopically produced bile juice istransported in the serum, and eventually processed in the native liverwithout affecting the host (our unpublished data). Similarly, in somecases, fluids and wastes might be successfully drained into thelymphatic vessels, allowing the ectopic graft to better survive. Inother cases, kidney products might accumulate inside the tubules,activating a positive loop of epithelial proliferation and vectorialfluid secretion, which eventually leads to cyst appearance. In otherwords, cyst formation inside the repopulated lymph node could share sometraits with multicystic dysplastic kidney (MCDK) and obstructivedysplasia (ORD), where urinary tract obstructive lesions cause urineretention in functioning nephrons and lead to cystogenesis [22].

Two main cysts were found in a repopulated lymph node (FIG. 26A). Cyst#1 was lined by a simple squamous epithelium, showing an apicalexpression of the water channel aquaporin-1 (AQP1) and absence ofsodium-potassium-chloride transporter 2 (NKCC2), indicating a possibleorigin from thin descending limbs of loop of Henle (FIG. 26B, left). Theepithelium was negative for BrdU indicating that cystic expansion hadalready ceased at the time of lymph node collection (FIG. 26B, left).The loop of Henle plays a role in the transport of ions and water,allowing production of urine. Accordingly, cyst #1 contained many ovalto round, rhomboid, parallelepiped, and amorphous urinary crystals, withmore or less sharply defined contours, some of them reaching 100 μm oflength (FIG. 26D, left). Cysts #1 also contained eosinophilic, Periodicacid-Schiff (PAS) positive, acidfuchsinophilic with Masson's trichrome(TRI), and red with picro-sirius red (PSR) staining proteinaceousmaterial, apart from amorphous fibers often containing a periodicbanding pattern, and rarely TRI positive (FIG. 26C, left). The presenceof urine in repopulated lymph nodes was confirmed by Blood Urea Nitrogen(BUN) test 16 weeks following kidney transplantation. While serum BUNlevels were not altered in transplanted mice as compared to controlmice, BUN levels were highly increased in lymph node fluid after kidneytransplantation and cyst formation (FIG. 26D, right). However, BUNlevels were not increased in repopulated lymph nodes where nomacroscopic cysts could be observed, further indicating that the timewindow of ectopic kidney maturation and degeneration differs among mice.Approximately, S-shapes bodies take 6 weeks to be converted into maturenephrons, and these nephrons can degenerate by the 12th week, as well asbe still healthy and functional at this stage.

Differently from cyst #1, cyst #2 was lined by a simple tall cuboidalepithelium, showing apical endocytic vacuoles and a PAS positive brushborder, indicating an origin from proximal convoluted tubule (FIG. 26B,right). Accordingly, the epithelium stained positive for AQP1 andnegative for aquaporin 2 (AQP2) (FIG. 26B, right). Moreover, it showedsome positivity for the BrdU marker, indicating cystic expansion processwas still active at the time of lymph node collection (FIG. 26B, right).The proximal convoluted tubule reabsorbs large molecules, such asproteins. Accordingly, cyst #2 contained pale eosinophilic, PASpositive, intensely acid-fuchsinophilic with TRI, and yellowish with PSRstaining round globules, ranging from 1 to 20 μm diameter, thought to beprotein globules (FIG. 26C, right). These structures likely are hyalinecasts covered with fat droplets. The accumulation of hyaline droplets isthe visible aspect of the damage to the glomerular capillary membrane,which leads to abnormal filtration and reabsorption of plasma proteins.

A very small cyst with all the features of cyst #2 was also identified(FIG. 26A-26C). A mix of GFP positive and negative cells lined this cyst(cyst #3), indicating a hybrid origin (FIG. 26E).

Structural glomerular alterations could be observed in thecyst-containing ectopic renal graft. Specifically, histological analysesoften revealed compressed tuft, in the center of the glomerulus,surrounded by a circumferential cellular crescent (H&E) (FIG. 26F,upper). There was a clear space between tuft and the crescent. A mildfocal thickening of glomerular basement membrane could be observed (PAS)(FIG. 26F, upper). Basement membrane thickening could be attributed toincreased collagen accumulation (TRI and PSR) (FIG. 26F, upper). Thecellular crescent contained some BrdU positive cells, indicating activeproliferation (FIG. 26F, upper). Expansion of the mesangial matrix inthese glomeruli was confirmed by intense staining for collagen IV (FIG.26F, upper). Hypercellularity within the glomerular tuft, obliteratingBowman's space was also observed (FIG. 26F, bottom). Hypercellularglomeruli can be due to immune cell infiltration. Close to theseglomeruli, swelling and vacuolization of proximal tubular cells leadingto narrowing of tubular lamina (osmotic nephrosis) were detected (FIG.26F, bottom). Taken together, although at a certain point, the ectopicgraft begins to degenerate, to our knowledge, our study shows the firstlong-term survival of metanephroi transplanted into an ectopic site.

Bone Marrow-Derived Host Cells Integrate into the Developing Tissue

On the basis of the results shown in FIGS. 3 and 4E, we hypothesizedthat kidney regeneration inside the lymph node could not only beattributable to transplanted kidney stem/progenitor cells, but couldalso be attributable to the combination of transplanted kidneystem/progenitor cells and stem cells of host origin such as bone marrow.

To investigate whether bone marrow contributes to ectopic kidneyorganogenesis into the lymph node, bone marrow chimeras were generatedas described in the Methods section. Engraftment was monitored by flowcytometric analysis of the peripheral blood 6 weeks after celltransplantation. All mice except one showed >75% of GFP+ leukocytes intheir blood (FIG. 27A). The mouse showing the lowest engraftment (BMT#5) died 8 weeks following transplantation and was therefore notincluded in the study. Mouse blood was also analyzed for differentmarkers of lymphocytes and granulocytes/monocytes. Gating strategy isindicated in FIG. 30A. Briefly, within the GFP+ CD45+ cell population,13.8%±5.1 were CD3+ cells; in turn this population comprised 61.6%±8.7CD4+ cells, and 8.7%±1.5 CD8+ cells. When looking at B-lymphocytes, wefound 40.2%±9.9 GFP+ CD45+ cells to be double reactive for CD19 and B220(FIG. 30B). Finally, 73.3%±15 of total Ly6G and Ly6C were GFP+ (FIG.30C).

Following 8 weeks from bone marrow transplantation, all mice receivedinjection of wild-type embryonic kidneys (FIG. 27B). Mice weresacrificed 6 or 10 weeks post kidney transplantation (FIG. 27B).Interestingly, bone marrow-derived collagen-producing cells wereincorporated in developed renal corpuscles 6 weeks after transplantation(FIG. 27C). Ectopic grafts were also stained for several markers ofcells of hematopoietic and nonhematopoietic origin, including CD45,CD106 (VCAM-1), CD3, CD4, CD8, CD45R/B220, Ly6C/G, and F4/80.Immunofluorescence analyses revealed that most GFP+ cells in theglomeruli were neither lymphocytes nor macrophages. Interestingly, bothGFP+ CD45− and GFP+ CD106+ cell subsets localized in ectopic glomeruli,suggesting the participation of bone marrow-derived mesenchymal stromalcells (MSCs) in ectopic kidney organogenesis (FIG. 27D). Moreover,glomerular GFP+ WT1+ podocytes were observed, suggesting that BMDCs cancontribute to ectopic podocyte regeneration. Nevertheless, it must notbe excluded that podocyte generation/replacement could also rely onresident renal cells. Nodular lesions were observed in 10 week-ectopicglomeruli (FIG. 31). Interestingly, cells with immunohistochemicalfeatures of parietal epithelial cells (PECs) could be detected at theglomerular tuft. These claudin-2+ cells shared the same location ofWT-1+ podocytes (FIG. 31). PECs lining the inner region of Bowman'scapsule have been shown to migrate onto the glomerular tuft anddifferentiate into podocytes [27]. Thus, in the lymph node-grown graft,PECs might transdifferentiate into podocytes. Cellular lesions alsoexpressed the PEC marker keratin-8 and the podocyte marker vimentin(FIG. 31). Interestingly, lesioned glomeruli showed a massive presenceof BMDCs. It remains to understand whether BMDCs contribute toregeneration of damaged glomeruli or facilitate extracellular matrixdeposition and as a consequence renal failure.

BMDCs did not contribute to vascularization of the ectopic graft, as noGFP+cells were incorporated in CD31+ vessels (FIG. 27D). Similarly,BMDCs did not contribute to the formation of kidney tubular structures(FIG. 27D, bottom), confirming the idea that tubule regeneration mainlyoccurs through survival of dedifferentiated epithelial cells whichproliferate and redifferentiate into mature functional epithelial cells[28].

Nephrectomy Accelerates Kidney Organogenesis and Degeneration

To assess whether the ectopic kidney tissue could proliferate inresponse to growth stimuli, we performed unilateral nephrectomy 12 daysafter embryonic kidney injection into the lymph node, and we added BrdUto the drinking water of the recipient mice as indicated in the Methodssection (FIG. 28A). The number of BrdU-positive nuclei per renalcross-section was significantly increased in contralateral kidneys ofleft nephrectomized animals (FIG. 28B). Ectopic kidneys isolated fromboth sham-operated and nephrectomized mice showed a variableproliferation rate. Importantly, grafts from mice undergoing leftnephrectomy, collected almost 4 weeks after kidney transplantation (26days, FIG. 28C), were comparable to the 12 week-ectopic grafts shown inFIGS. 3-4. Either grafts comprising fully mature and apparently healthynephrons or grafts comprising enlarged and swollen glomeruli were infact observed. Thus, although the stimulus that results in compensatoryrenal growth following reduction of renal mass is dispensable for kidneyorganogenesis into the lymph node, nephrectomy accelerates ectopickidney maturation. To our opinion, this finding further reinforces ourhypothesis that ectopic kidney degeneration is a consequence offunctionality rather than a result of aberrant kidney development.Moreover, since native renal tissue needs to be removed to successfullygrow metanephroi in the omentum [4], our findings suggest that the lymphnode could provide a much more better site than omentum for ectopickidney organogenesis.

Discussion

In contrast to lower vertebrates, in mammals, nephrogenesis is limitedto gestation or early post-natal life. Although the adult kidney cannotmake new nephrons, it can regenerate and recover in some circumstances.Indeed, tubular regenerative capacity widely changes going from acutekidney injury (AKI) to chronic kidney disease (CKD), as acute renalinsults are handled with successful regeneration, while chronic injurieslead to ineffective or even more damaging cellular responses [29]. Moreprecisely, nephron tubule epithelium is regenerated after AKI, while inthe setting of CKD, tubular damage is not repaired, and this isaccompanied by a sustained inflammatory response and activation ofmyofibroblasts, that eventually results in interstitial fibrosis,tubular atrophy, and nephron loss. Although many theories exist onkidney repair, the existence of an intratubular cell source fuellingnephron tubule epithelium regeneration after AKI is gaining consensusamongst researchers [30].

A number of experimental evidences have indicated transplantation ofembryonic kidney as a potential method for augmentation of kidneyfunction [4, 14-17]. However, various hurdles remain before a clinicaltherapy can become a reality. Cell culture techniques to produce renalorganoids starting from mouse embryonic kidney precursors have beendescribed, but several experimental attempts to develop functionalglomeruli have failed, because the avascular in vitro environment is notpermissible for glomerulogenesis.

The current studies indicate that the lymph node might be considered asa unique niche to grow several tissues. When embryonic kidneys weretransplanted into the lymph node, blood vessels integrated into theglomeruli. Vascularization is likely attributable to migration andproliferation of resident endothelial cells, and does not involve BMDCs.However, both bone marrow hematopoietic and stromal cells were found inthe ectopic kidney graft. These cells contributed to mesangial cells andpodocyte regeneration. Not only did the lymph node furnish thedeveloping tissue with host cells, but also provided it with growth andhomeostatic signals, since a decrease in native renal mass could pushmaturation of the ectopic graft. In conclusion, the present studyprovides evidence that ectopic kidney inside the lymph node can sense astimulus and appropriately respond. This system can also be used tovalidate in vivo the differentiation potential of candidate cells inregenerative nephrology, including ES or iPS.

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EXAMPLE 5: THE MOUSE LYMPH NODE; A TOOL FOR STUDYING FUNCTIONALMATURATION Summary

Our results indicate that it is possible to obtain postnatal developmentstages for several mid-embryonic tissues including brain, thymus, lung,stomach, and intestine, inside the lymph node (LN). We believe the LNmight provide a unique tool to track and monitor stem cell behavior invivo, in a location far from the native environment, but stillresponsive to physiologic and homeostatic signals.

Methods Tissue Collection and Transplantation

Embryonic day (E) 14.5 to 15.5 tissues were retrieved from timedpregnant GFP+ C57BL/6 black mice under a dissecting microscope (embryoswere considered 0.5 days old when the vaginal plug was detected in themorning). All tissue were chopped in PBS and kept on ice untilinjection. For lymph node transplantation, recipient mice (wild-typeC57BL/6 black mice, n=51) were anesthetized with 1-3% isoflurane. Asmall incision was made in the abdomen to expose jejunal lymph nodes. A1000 μL threaded plunger syringe (Hamilton, 81341) with a removableneedle (gauge 20) was used to slowly inject tissue fragments into asingle lymph node. Light cauterization was used to seal the opening. Thewound was then closed with surgical sutures. Ketoprofen (2 mg/kg, IM)treatment for postoperative pain relief was initiated right aftersurgery and continued for 2 additional consecutive days. Mice were bredand housed in the Division of Laboratory Animal Resources facility atthe University of Pittsburgh Center for Biotechnology andBioengineering. Experimental protocols followed US National Institutesof Health guidelines for animal care and were approved by theInstitutional Animal Care and Use Committee at the University ofPittsburgh.

Histology and Immunofluorescence/Immunohistochemistry

Repopulated jejunal lymph nodes were fixed 2 hours in 4% PFA, andembedded in Optimal Cutting Temperature (OCT) following infiltrationwith 30% sucrose overnight. Sections were stained with antibodiesagainst GFAPδ (Bioss, bs-11016R), GFP (Abcam, ab6556), Keratin-8 (DSHB,TROMA-1), Keratin-5 (Covance, PRB-160P), GFP (Abcam, ab6556), CD31 (BD,550274), CD105 (BD, 550546), ER-TR7 (Abcam, ab51824), MUCSAC (Abcam,ab79082), β-catenin (CST, 8480), MUC2 (SCT, sc-15334), or chromogranin A(SCT, sc-18232). Alexa Fluor 594 (Invitrogen) secondary antibodies werethen used. Nuclei were counterstained using Hoechst. Donor organs wereembedded in paraffin and stained with hematoxylin and eosin (H&E) asdescribed elsewhere.

RNA Extraction, cDNA Synthesis, RT-PCR

Total RNA was isolated from tissues stored in RNAlater® reagent (QIAGEN)using RNeasy Mini kit (QIAGEN), according to the manufacturer'sinstructions. Potentially contaminating genomic DNA was digested usingDNase (QIAGEN). cDNA was synthesized using the iScript™ ReverseTranscription Supermix for RT-qPCR (Bio-Rad). PCR was performed usingthe iTaq DNA Polymerase kit (Bio-Rad). GAPDH transcript levels served asthe housekeeping control target. Sequences of primers were as follows:GM-CSF, Fwd, 5′-TTCCTGGGCATTGTGGTCT-3′ (SEQ ID NO: 12), Rev,5′-TGGATTCAGAGCTGGCCTGG-3′ (SEQ ID NO: 13); GAPDH, Fwd,5′-GGCATCCTGGGCTACACTGA-3′ (SEQ ID NO: 10), Rev,5′-GGAGTGGGTGTCGCTGTTG-3′ (SEQ ID NO: 11). For GM-CSF, PCR mixtures weresubjected to different numbers of amplification cycles; 25 cycles wereeventually chosen to quantify gene expression.

Flow Cytometry

Whole blood was collected in K2EDTA collection tubes (Terumo Medical).One hundred microliters of blood was added to coldfluorescence-activated cell sorting (FACS) tubes. Antibodies were addedat a dilution of 1/50 in blood and mixed by gentle pipetting. Antibodiesused were as follows: PerCP Cy5.5 CD45 (BD, 550994), PECD11b (BD,553311), and APC Ly6G−Ly6C (BD, 553129). Reactions were incubated in thedark in an ice slurry bath for one hour. Three milliliters of Red BloodCell Lysing Buffer (Sigma) was added to each tube, lightly vortexed andincubated for an additional 15 min. Two milliliters of flow buffer (2%FBS in HBSS) was added to the tubes, mixed and centrifuged at 500 g for5 min. The supernatant was aspirated. The red blood cell lysis andcentrifuge were repeated as described. The final cell pellet wasresuspended in 400 μl of flow buffer with Sytox Blue dye. Cells wereanalyzed using a Miltenyi MACSQuant and FlowJo software (Tree Star).

Statistical Analysis

Data are presented as means±SD. Statistical analysis was performed usingStudent's t test (p<0.05 was considered significant).

Results The Lymph Node is a Permissive Site for Tissue Organogenesis

We first investigated the ability of lymph node to support engraftmentof several mouse mid-embryonic tissues including brain, thyroid, thymus,lung, heart, stomach, intestine, liver, and adrenal gland. Tissues wereharvested from E14.5/E.15.5 GFP transgenic mice, minced, and injecteddirectly into a single jejunal lymph node of adult wild-type mice (FIG.32A). Following 3 weeks, recipient mice were sacrificed, lymph nodescollected, and histologically examined. As shown in FIG. 32B,transplants were variably prone to engraft into the lymph node. Whilethe brain showed the highest ability of repopulating the lymph node, thethyroid was unable to engraft in. Although in some cases heart, liverand adrenal gland engrafted, their grafts were very small. We thereforefocused our attention on brain, thymus, lung, stomach, and intestine.

Astrogenesis in the Developing Ectopic Brain

In the developing mouse brain, glial fibrillary acid protein delta(GFAPδ) starts being expressed at E18. Accordingly, we found very lowGFAPδ expression in E14.5-E.15.5 mouse brain (FIG. 32C2, upper).Importantly, when transplanted into the LN, E14.5/15.5 brain engrafted(FIG. 32C1) and maturated (FIG. 32C2, lower), as indicated by presenceof GFAPδ⁺ cells with complex branching and extended processes,indicative of mature astrocytes.

Maturation of the Thymic Epithelium in an Ectopic Site: Contribution ofthe Host in the Generation of the Thymic Cortex

Immune cell percentages were monitored after thymus transplantation.Gating strategy is indicated in FIG. 33A. The CD11b+/Ly6G−Ly6C−/low andCD11b+/Ly6G−Ly6Cint cell populations (mainly comprising immature myeloidcells) were dramatically increased following 3 weeks from thymustransplantation, to the detriment of the CD11b+/Ly6G−Ly6Chigh cellpopulation (mainly comprising mature myeloid cells, such asmetamyelocytes/granulocytes) (FIG. 33B-33D). All cell populationsreturned to control levels within 21 weeks (FIG. 33B-33D). Slightchanges were also observed within the lymphocyte population. Overall,these changes reflect functionality of the ectopic graft. The strikingeffect on the CD11/Ly6GLy6C phenotype possibly indicates maturation ofthe ectopic thymic epithelium, and enhanced transcription of thegranulocyte-macrophage colony-stimulating factor (GMCSF). Production ofthis cytokine eventually results in expansion of immature myeloid cells,and as a consequence, in the accumulation of granulocyte/macrophageprogenitors. During thymic ontogeny, GM-CSF mRNA reaches its peak levelbetween E19-20; before this stage, it is undetectable during the earlyamplification cycles of a semi-quantitative PCR (5, 6). We thereforeanalyzed GM-CSF mRNA levels in the ectopic thymus, as compared to themid-embryonic thymus used for transplantation. While levels of GMCSFmRNA were very low in E14.5/E.15.5 mouse thymus, GM-CSF mRNA was clearlypresent in 6-week ectopic grafts, as well as in 21-week grafts, but to aless extent than in the adult thymus (FIG. 34A). Ectopic grafts wellvaried on size (FIG. 34B, lower). While embryonic thymus had a uniformparenchyma with light staining (FIG. 34B, upper right), 21 week ectopicthymus comprised a meshwork of epithelial cells reactive to antibodiesagainst keratin 8 or keratin 5, indicating a cortical or a medullaryidentity, respectively (FIG. 34C). Nevertheless, a clearcorticomedullary compartimentalization was never observed.Interestingly, most of keratin 8+ cells were of host origin, while mostof keratin 5 positive cells were of donor origin (FIG. 34C, right).Ectopic thymic grafts also showed chimeric blood vessels (FIG. 34C,lower right). These vessels were reactive for CD105, a marker forneoangiogenesis (FIG. 34C, lower right).

Overall, our findings reveal that the lymph node might be exploited tounderstand how and when cortical and medullary lineages diverge. Ourresults indicate cortical (cTEC) and medullary (mTEC) thymic epithelialprogenitors might follow independent differentiation pathways.

Presence of Terminally Differentiated, Mucus-Producing Cells in EctopicLung, Stomach and Intestine Tissues

Embryonic lung fragments arranged in lobe-like structures into the lymphnode, and showed sign of differentiation from a pseudoglandular to mixedsaccular/alveolar morphology 3 weeks after transplantation. Importantly,ectopic lung comprised a glandular epithelium with MUC5AC-producinggoblet cells 10 weeks after transplantation, indicating that it ispossible to achieve postnatal stages of mouse lung development insidethe lymph node (FIG. 35A).

Similarly, mid-embryonic stomach fragments well engrafted and expandedinside the jejunal lymph node. This ectopic stomach also comprisedMUCSAC-producing goblet cells 3 weeks after transplantation (FIG. 35B).

While intestinal development occurs quite early during mammalianembryogenesis, the intestinal maturation takes place during thepost-embryonic period. Crypt-like structures developed followinginjection of intestinal fragments into lymph nodes. Membrane-localizedβ-catenin was detected all along these crypts. Activity of theWnt/β-catenin pathway is required for the emergence of secretory celltypes in the intestinal epithelium (16). Since both MUC2-reactivegoblet-like cells and chromogranin A (CgA)-producing enteroendocrinecells could be detected in intestinal grafts 3 weeks aftertransplantation, our results likely indicate that Wnt signals wereactive and supported intestinal epithelium terminal differentiationinside the lymph node (FIG. 4C).

Discussion

In vivo, stem cells reside in a highly specialized three-dimensional(3D) structure, the socalled niche (17). Not only does the nichepreserve the stem cell pool, but also promotes progenitor cell expansionand mobilization. Reproducing this dynamic and complex microenvironmentin culture is challenging, either because the mechanisms that controlstem cell fate in vivo have not yet been fully elucidated, or because ofethical and technical issues. The current study indicates that the lymphnode mimic the physiological environment of transplanred tissue andpromotes the vascularization of the transplanted tissue. Lymph nodeshave ready access to the bloodstream, and can therefore foster cellgrowth by providing nutrients as well as hormones and growth factors.Accordingly, we here showed the ability of mouse lymph node insupporting organogenesis of different tissues including brain, thymus,lung, stomach and intestine. Lymph node-grown tissues more closelyrecapitulate in vivo phenotypes under physiological conditions than anyother culture system.

References for Example 5

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EXAMPLE 6: ENGINEERING AN ECTOPIC THYMUS IN THE LYMPH NODE TO INDUCECENTRAL TOLERANCE AND ALLOGRAFT ACCEPTANCE Summary

Allogeneic transplant rejection is one of the major problems plaguingthe field of organ transplants today. In the current study, wehypothesized that by conditioning transplant recipients with thymiccells immune-matched to the donor, we will be able to induce long-termtolerance of a subsequent allograft. We were able to achieve long-termallograft acceptance in mice receiving thymic transplants. The allograftacceptance maybe mediated by the increased Treg induction associatedwith cross-talk between the two thymuses

Methods

We utilized the FAH−/− mouse model on a 129sv background for ourstudies. As shown in FIG. 37, We transplanted Balb/c GFP thymuses intothe lymph nodes of FAH−/− mice that were transiently immunosuppressed tofacilitate acceptance of the thymic grafts. About 6 weeks after thethymus transplant, the animals were further challenged withdonor-matched skin grafts or hepatocyte transfer. The recipients werethen monitored for long-term allograft acceptance. In addition, weassessed induction of central tolerance and ectopic-native thymuscross-talk by performing mixed lymphocyte reaction assays (MLR),immunostaining of native and ectopic thymus, as well as thymic dendriticcells (tDC)-T cell co-cultures to assess Treg generation.

Results

As shown in FIG. 38, donor thymus tissue successfully engraftd intorecipient lymph node. As shown in FIG. 39, mice with thymus transplantsdemonstrated long-term acceptance of allografts (skin grafts as well ashepatocyte transfers). Furthermore, as observed in the mixed lymphocytereaction (MLR) assays, these mice were specifically tolerized to theBalb/c strain, but were reactive against the C57BL/6 strain (FIG. 40).We also observed migration of cells (mostly tDCs) from the ectopic tothe native thymus (FIG. 41). Analysis of tDC-T cell co-cultures revealedthat the migrating DCs from the ectopic as well as native thymus wereinstrumental in generating the increased numbers of Tregs observed (FIG.42).

Conclusion

We were able to induce long-term acceptance of thymus-matched allograftsin immmunocompetant mice. Thymus transplants induced acceptance ofallogeneic hepatocytes and rescue of liver function in the 129.Fah−/−mouse model. Migration of antigen-presenting cells to induce Tregs, andcross-talk between the two thymuses appears to be important forinduction of central tolerance and allograft acceptance.

Various references are cited herein, the contents of which are herebyincorporated by reference in their entireties.

1-17. (canceled)
 18. A method of evaluating maturation of an ectopicgraft epithelium, the method comprising: (a) obtaining RNA from theectopic graft epithelium; (b) reverse transcribing the RNA from theectopic graft epithelium to generate cDNA; and (c) performing apolymerase chain reaction (PCR) to quantify an amount of GM-CSFexpressed in the ectopic graft epithelium; thereby evaluating thematuration of the ectopic graft epithelium.
 19. The method of claim 18,wherein the RNA is obtained 21 weeks after the ectopic graft isadministered to a subject.
 20. The method of claim 18, wherein the PCRcomprises about 25 reaction cycles.
 21. The method of claim 18, whereinthe PCR comprises a first primer comprising the nucleotide sequence ofSEQ ID NO: 12 and a second primer comprising the nucleotide sequence ofSEQ ID NO:
 13. 22. The method of claim 18, further comprising comparingthe amount of GM-CSF expressed in the ectopic graft epithelium to anamount of GM-CSF expressed in an E14.5/E.15.5 mouse thymus.
 23. Themethod of claim 22, further comprising evaluating expression of keratin8 in the ectopic graft epithelium and the E14.5/E.15.5 mouse thymus viaimmunofluorescence staining and fluorescence microscopy.
 24. A nucleicacid comprising the nucleotide sequence of any one of SEQ ID NOs: 1-11.25. A method, comprising: (i) backcrossing 129sv Fah^(−/−) mice for morethan eight generations with C57BL/6 mice, thereby generating a C57BL/6Fah^(−/−) mouse, and (ii) injecting a dye solution into a footpad of theC57BL/6 Fah^(−/−) mouse.
 26. The method of claim 25, wherein the dyesolution comprises 3% evans blue solution.
 27. The method of claim 25,wherein the dye solution is injected intradermally into the footpad of ahindlimb of the C57BL/6 Fah^(−/−) mouse.
 28. The method of claim 27,further comprising visualizing a popliteal lymph node in the hindlimb ofthe C57BL/6 Fah^(−/−) mouse.